Methods for Generation for Pluripotent and Multipotent Cells

ABSTRACT

This disclosure relates to methods of producing induced pluripotent (iPS), multipotent, and/or lineage-committed stem cells from differentiated cells, maintaining iPS, multipotent, and/or lineage-committed cells in culture, and re-differentiating the iPS and multipotent stem cells into any desired lineage-committed cell type.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/390,134, filed Oct. 2, 2014, which is a national stage applicationunder 35 U.S.C. 371 and claims the benefit of PCT Application No.PCT/US2013/035838 having an international filing date of Apr. 9, 2013,which designated the United States, which PCT application claimed thebenefit of U.S. Provisional Application No. 61/621,994, filed Apr. 9,2012, and U.S. Provisional Application No. 61/735,701, filed Dec. 11,2012, the disclosure of each of which are incorporated herein byreference in their entirety.

FIELD

This disclosure relates to methods of producing induced pluripotent stem(iPS), multipotent, and/or lineage-committed cells from differentiatedcells, maintaining iPS, multipotent, and/or lineage-committed cells inculture, and re-differentiating the iPS and/or multipotent cells intoany desired lineage-committed cell type.

BACKGROUND

A primary goal of regenerative medicine is replacement of diseased ordamaged cells and tissues. Abundant and safe sources of multipotent orpluripotent stem cells are necessary to further this goal. Embryonicstem (ES) cell lines are available for possible regenerative medicineapplications, but challenges remain for their use, including possibleimmune rejection by a receiving patient (reviewed in Yabut et al., Aging3(5):494-508, 2011). In recent years, induced pluripotence indifferentiated cells has been explored as an alternative to ES cells(reviewed in Ebben et al., World Neurosurg. 76(3-4):270-275, 2011). Itwas discovered that expression of just four stem cell transcriptionfactor genes (c-Myc, Sox2, Klf4, and Oct4) can de-differentiate andinduce pluripotence in cells grown under particular culture conditions(e.g. in the absence of serum) (WO 2012/012708; and Takahashi el al.,Cell. 126: 663-676, 2006). Among other benefits, such inducedpluripotent stem (iPS) cells might be generated from a potentialpatient's own cells, thereby minimizing adverse immunoreactivity uponintroduction of pluripotent or newly-differentiated cells to thepatient.

iPS cells are currently produced by transforming cells with viral orother constitutive expression vectors encoding the four stem celltranscription factor genes. Among these, the over-expression of c-Myc isof particular concern because sustained Myc expression can result inmalignant transformation. Furthermore, any of these vectors canpermanently integrate into the cellular genome at sites that activateoncogenes or disrupt tumor suppressor genes. Current efforts in the stemcell field to produce iPS cells without the risk of malignanttransformation involve identification of small molecules to induceindividual stem cell genes (c-Myc, Sox2, Klf4, and Oct 4), with the goalof designing a mixture of several small molecules that together canproduce iPS cells. But to date, no single agent has been identified thatcan be used to produce iPS cells. Thus, a continuing need exists toidentify agents that can produce iPS cells, without the need forplasmid- or retroviral-mediated expression of individual stemcell-inducing genes.

SUMMARY

Described herein are the surprising observations that blockade ofsignaling by the cellular receptor CD47 results in significantlyincreased cellular lifespan and expansion of lineage-committed ordifferentiated cells in culture, and when such cells are grown inappropriate media (such as serum-free media), production of multipotentor iPS cells. These cellular phenotypes are associated with increasedexpression of the transcription and cell proliferation factor c-Myc, andincreased expression of the hallmark stem cell-inducing transcriptionfactors Sox2, Klf4, and Oct4. In appropriate culture media, themultipotent or iPS cells can then be differentiated into desired celltypes, which can be expanded and maintained in culture by transient,intermittent, or continued CD47 blockade.

Based upon these observations, methods are enabled and described hereinfor generating and/or expanding lineage-committed stem cells,multipotent stem cells, and/or iPS cells from lineage-committed ordifferentiated cells by CD47 blockade. CD47 signaling blockade can beachieved in any way or with any agent that inhibits CD47 expression onthe cell surface, or that blocks CD47 intracellular signaling, such asby blocking the binding of CD47 ligands, including blocking binding ofthe matricellular protein thrombospondin-1 (TSP1). In particularembodiments of the disclosed methods. CD47 blockade can be achieved bycontacting cells with one or more TSP1-derived peptides, anti-CD47 oranti-TSP1 antibodies, anti-CD47 or anti-TSP1 antisense oligonucleotidesor morpholinos or other stabilized nucleic acid molecules. These andother methods of blocking CD47 signaling are described in detail in U.S.Patent Publications No. US 2010/0092467 and US 2011/0135641, which arehereby incorporated by reference in their entirety. In otherembodiments, CD47 signaling blockade can be achieved by contactingCD47-expressing cells with a chemical agent (such as a small moleculeagent) that binds to CD47 or TSP1 and blocks or reduces CD47-signaling.

In particular embodiments, the described methods include obtainingprimary cells (such as lineage-committed (differentiated) cells) from ananimal or subject and contacting the obtained cells with an agent thatcan block CD47 signaling. Multipotent or pluripotent stem cells areproduced from the CD47-blocked cells when the blocked cells are culturedin appropriate culture media, which in particular embodiments includesserum-free medium.

Also described herein are methods of maintaining stem cells in ade-differentiated state capable of self-renewing proliferation bycontinued exposure of the cells to an agent that blocks CD47 signaling.The de-differentiated state is maintained as long as the cells arecultured in appropriate media and exposed to a CD47 blocking agent. Insome embodiments transient exposure to a CD47 blocking agent issufficient to induce this de-differentiated state resulting in cellscapable of self-renewing proliferation.

Further described herein are methods of producing a desireddifferentiated cell type from a previously lineage-committed cell type.Desired cell types can be produced by generating multipotent or iPScells using a CD47 blocking agent as described above, and then removingthe CD47 blocking agent from the iPS cells, while also culturing the iPScells in medium containing appropriate differentiating factors known tothose of ordinary skill in the art. In some examples, thenewly-differentiated cells can be immortalized for storage byre-exposure to a CD47 blocking agent. Such cells will maintain theirdifferentiated state in the appropriate media, such as serum-containingmedia.

Additionally described herein are iPS cells produced by the describedmethods, and lineage-committed cells differentiated from the producediPS cells.

Also described herein are methods to employ CD47 blockers tocontinuously expand lineage-committed stem cells or iPS cells from asmall amount of donor tissue or cell aspirate that can later bere-administered to that donor.

One of ordinary skill in the art will appreciate that the ability togenerate and maintain a ready supply of multipotent or iPS cells fromwhich lineage-committed cells can be produced using a single definedagent will have significant benefits in the field of regenerativemedicine. This approach will also greatly expand the potentialapplications of autologous stem cell therapy, including applicationswhere genetic defects are corrected ex vivo before re-administering theexpanded cells to an individual suffering from an inherited or acquiredgenetic defect.

The foregoing and other features will become more apparent from thefollowing detailed description, which proceeds with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show enhanced proliferation and decreased senescence ofCD47-null murine endothelial cells. FIG. 1A is a graph of a3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay for cell survival and growth over 72 hours expressed as % ofday 0 values at the indicated plating densities of first passage WT andCD47 null cells. FIG. 1B is a graph of a 5-bromo-2′-deoxyuridine (BrdU)assay for DNA synthesis. FIG. 1C is a graph of percentage ofsenescence-associated β-galactosidase (β-gal) expression at passage 3(*p<0.05. **p<0.01).

FIGS. 2A-2F show CD47 signaling inhibits c-Myc and additional stem celltranscription factor expression in vitro and in vivo. FIG. 2A is a chartshowing expression of genes associated with cell immortalization in WTand CD47 null cells. FIG. 2B is a graph of c-Myc mRNA levels in lungendothelial cells of CD47 null and WT mice. FIG. 2C is a digital imageof a Western blot showing c-Myc levels in WT and CD47 null mouse lungendothelial cells. CD47 limits c-Myc protein levels. FIG. 2D is a graphshowing mRNA expression levels of stem cell transcription factors in WTand CD47 null lung endothelial cells. From left to right, bars indicateKlf4, Sox2. Oct4, and Nestin mRNA levels for WT and CD47 null cells.FIG. 2E is a pair of digital images showing detection of c-Mycexpression by immunofluorescence in WT and CD47-null endothelial cells.FIG. 2F is a pair of panels showing flow cytometric analysis of c-Mycexpression in WT and CD47-null endothelial cells (*p<0.05, **p<0.01).

FIGS. 3A-3K show stem cell and differentiation marker expression inCD47-null endothelial cells and embryoid bodies induced by serum-freemedium. FIG. 3A is a series of digital images showing CD47-nullendothelial cells stained using the indicated antibodies and DAPI. FIG.3B is a series of digital images showing typical appearance of embryoidbody (EB)-like clusters photographed under phase contrast or stainedusing the indicated antibodies and DAPI. FIG. 3C is a plot showinganalysis of CD14 and CD11c in CD47-null endothelial cells using flowcytometry. FIG. 3D is a plot showing Sca-1 expression in CD47-nullendothelial cells. FIG. 3E is a series of digital images of Westernblots for protein expression of stem cell transcription factors fromcultured WT or CD47 null endothelial cells (in EGM2 medium). FIG. 3F isa pair of panels showing protein expression of Oct4 by flow cytometry incultured WT or CD47-null endothelial cells in EGM2 medium. FIG. 3G is apair of panels showing asymmetric cell division in second passage WT andCD47-null endothelial cells equilibrium labeled with bromodeoxyuridine(BrdU) and chased for one cell division. Asymmetric division was scoredby counting BrdU⁺/DAPI⁺ nuclei adjacent to BrdU⁻/DAPI⁻ nuclei. FIG. 3Hshows flow cytometric analysis of c-Myc expression in CD47-null cellsdissociated from EB-like clusters. FIG. 3I shows detection of asymmetriccell division in cells from CD47-null EB-like clusters equilibriumlabeled with BrdU, and then chased for two cell divisions without BrdU.Top left, DAPI; middle left, phalloidin; bottom left, BrdU; bottom rightcombined image. FIG. 3I is a pair of digital images showing morphologyof CD47-null EB-like clusters (left) and V6.5 ES cells (right) growingin ES medium with LIF. The V6.5 culture also contains an MEF feederlayer. FIG. 3K is a series of digital images showing CD47-null EB-likeclusters (center) and V6.5 ES cells (left) cultured as in FIG. 3J andCD47-null endothelial cells in endothelial growth medium (right) stainedusing the indicated antibodies and DAPI.

FIGS. 4A-F show differentiation of CD47-null EB-like clusters. FIG. 4Ais a series of digital images of EB-like clusters cultured in RPMIcomplete medium for 6 days and then transferred to lineage-specificmedia for 36 hours and stained with smooth muscle actin antibody todetect mesodermal cells. FIG. 4B is a series of digital images ofdifferentiated EB-like clusters stained with the ectoderm neural markersglial fibrillary acidic protein (GFAP) and neuron-specific beta IIItubulin (TUJI). FIG. 4C is a series of digital images of differentiatedEB-like clusters stained with anti-α-fetoprotein (AFP) to detectectodermal cells. In all panels DAPI was used to visualize nuclei. FIGS.4D-F show expansion of a single clone isolated from a CD47-null EB-likecluster expanded in serum-free medium and then differentiated in therespective lineage-specific medium for 7 days and stained for SMA (FIG.4D). TUJI (FIG. 4E), or AFP (FIG. 4F).

FIGS. 5A-H shows that CD47 regulates stem cell transcription factors invivo. FIG. 5A is a graph showing c-Myc mRNA from lung, kidney, liver,brain and spleen of WT and CD47-null mice. FIG. 5B is a graph showingc-Myc mRNA levels in purified splenic cell populations from WT (leftbars) and CD47 null (right bars) mice. FIG. 5C is a graph showing mRNAexpression levels of the indicated genes in spleen from WT (left bars)and CD47-null (right bars) mice. FIG. 5D is a graph showing mRNAexpression levels of the indicated genes in lung from WT (left bars) andCD47-null (right bars) mice. (For panels A-D, *p<0.05, **p<0.01). FIGS.SE-H are a series of digital images showing increased frequency of Sox2expressing cells in tissues from CD47-null mice. The alveolar (Alv)regions of lung tissues from WT (FIG. 5E) generally lack Sox2-positivecells, whereas CD47-null lung shows more positive cells (FIG. 5F). Incontrast, similar uniform Sox2 staining was observed in bronchiolarepithelium (BrEp) from WT and null mice (FIGS. 5E and F), consistentwith its previously reported expression in Clara cells (Tompkins et al.PLoS One 4: e8248, 2009). Paraffin embedded sections of representativespleen tissues from WT (FIG. 5G) and CD47−/− (FIG. 5H) mice were stainedwith a specific antibody to Sox2. Sections were examined under lightmicroscopy showing subcapsular (CP), red pulp (RP) and white pulp (WP)staining.

FIGS. 6A-6F show that CD47 expression regulates c-Myc and stem celltranscription factor expression. FIG. 6A is a graph showing morpholinoknockdown of CD47 (CD47-MO) in WT lung endothelial cells increases c-MycmRNA expression, but a control mismatched morpholino (mis-MO) does not.FIG. 6B is a graph showing in vivo morpholino knockdown of CD47 elevatesc-Myc, Oct4, and Sox2 mRNA at 48 hours in mouse spleen (left bars, WT:right bars, CD47-MO). FIG. 6C is a graph showing CD47 re-expression inCD47-null murine endothelial cells suppresses cell growth (left bars)unless c-Myc expression is sustained (CD47+MYC, right bars). FIG. 6D isa graph showing CD47 re-expression in CD47 null endothelial cells altersc-Myc expression. FIG. 6E is a graph showing expression levels oftransfected human CD47. FIG. 6F is a graph showing re-expression of CD47with an internal FLAG tag (CD47-FLAG) and c-Myc alters mRNA expressionof stem cell transcription factors (*p<0.05, **p<0.01). For eachcondition, from left to right, bars indicate Klf4, Nestin, Oct4, andSox2.

FIGS. 7A-71 show regulation of c-Myc and stem cell transcription factorsby CD47 ligation. FIG. 7A is a graph showing c-Myc mRNA in Jurkat (JK)and CD47-deficient JinB8 T cells (JIN). FIG. 7B is a graph showingtime-dependence for regulation of c-Myc mRNA expression by the CD47ligand thrombospondin-1 (TSP1). Jurkat cells were treated with 2.2 nMthrombospondin-1 for the indicated times before isolating RNA andassessing c-Myc mRNA by real time PCR normalized to β2-microglobulinmRNA and expressed as ratio to normalized c-Myc levels in control cellsat the corresponding time points. FIG. 7C is a graph showing TSP1effects on c-Myc mRNA in WT Jurkat (diamonds) and CD47-deficient Tlymphoma cells (squares). FIG. 7D is a graph showing CD47 re-expressionin JinB8 cells (JIN+CD47-V5) alters expression of c-Myc compared with WTJurkat cells. FIG. 7E is a graph showing effects of CD47-binding peptide7N3 and control peptide 604 on c-Myc mRNA in Jurkat T cells. FIGS. 7Fand G are graphs showing mRNA levels in TSP1-null vs. WT lung (FIG. 7F)and spleen. (FIG. 7G). For each condition, from left to right, barsindicate c-Myc. Sox2, Oct4. Nestin, and Klf4. FIG. 7H is a graph showingCD47 over-expression in Rat1 fibroblasts (right bars) and B16 melanomacells (left bars) does not suppress growth. FIG. 7I is a graph showingderegulation of translocated c-Myc in Raji Burkitt's lymphoma cellsprevents growth regulation by CD47 over-expression. (*p<0.05. **p<0.01).

FIGS. 8A-D show continuous propagation of WT and CD47-null mouse lungendothelial cells. FIG. 8A is a series of digital images of WT (top) orCD47-null (bottom) cultures at 7 days after each passage (P1-P3). FIG.8B is a series of digital images of WT cells at passage 2 (left), whichshowed a flattened morphology characteristic of senescent cells, whileCD47-null cells (right) maintained a typical endothelial morphology. Thegrowth of both WT and CD47 null lung endothelial cells slowed afterpassages 3-5. WT cells grew very slowly and became stationary senescentcells. On the other hand, CD47 null cells initially flattened butresumed growth within 2-3 weeks. CD47 null cells restarted growth ascolonies of well differentiated endothelial cells that maintainedextensive cell-cell contact (cobblestone morphology) and requiredpassage twice a week. Independent isolates of CD47 null endothelialcells reproducibly maintained their growth and morphology for at least 6months. WT cells never resumed growth. FIG. 8C is a pair of digitalimages of mouse lung endothelial cells from WT and thrombospondin-1 nullmice. Equal numbers of WT and thrombospondin-1 null murine lungendothelial cells were plated at the indicated passage numbers. Aftergrowth in EGM medium plus 0.5% FBS, viable cells were quantified bytrypsinization, centrifugation, and counting on a hemocytometer in thepresence of Trypan blue (FIG. 8D).

FIG. 9A is a series of digital images of formation of embryoid bodies byCD47-null endothelial cells transferred into serum free neural basalmedium. Sequential photographs of a representative culture are shown.FIG. 9B is a series of digital images showing selective formation ofEB-like clusters by passage 2 CD47-null endothelial cells in serum-freemedium. Adherent cells (left) and non-adherent cells (right) were imaged36 hours after transfer into serum-free medium. Nascent non-adherentEB-like clusters were abundant in the CD47-null culture, but only oneloose cluster of cells was observed in the WT control. The latter cellsdid not survive at later times.

FIGS. 10A-H are a series of digital images of WT (FIGS. 10A-D) andCD47-null (FIGS. 10E-H) mouse lung endothelial cells cultured in EGM2medium and then transferred to serum-free medium to induce embryoidbodies and stained for pluripotent stem cell markers. Alkalinephosphatase activity (dark staining) was observed in embryoid body cellsderived from CD47-null endothelial cells (FIGS. 10F-G), whereas noalkaline phosphatase activity was observed in WT cells, which failed toform EBs (FIGS. 10B-D).

FIGS. 11A-G show morphological and biochemical analysis ofdifferentiated embryoid bodies derived from CD47-null cells for 10-15days. FIGS. 11A-B show differentiated EBs under bright field and phasecontrast illumination, respectively. Representative H&E stained sectionshows morphological evidence for ectodermal, mesodermal, and endodermaldifferentiation (FIGS. 11C-F). A 5 μm formalin fixed paraffin embeddeddifferentiated embryoid body stained with H&E (4× objective, FIG. 11C)indicates the presence of all three germ cells layers: cuboidalendodermal epithelium with slightly atypical nuclei (H&E 40× objective,FIG. 11D), mesoderm or primitive mesenchyme with oval/fusiforme nucleiembedded in a myxoid matrix (H&E 40× objective. FIG. 11E). Some of thecells (arrows) contain eosinophilic amorphous material. Numerousapoptotic bodies are also seen (H&E 40×, FIG. 11E). FIG. 11F also showspresumptive ectoderm with pluristratified monotonous, basophilic nucleimimicking primitive neuroectoderm (H&E 20×, FIG. 11F). FIG. 11G is aWestern blot showing biochemical analysis of embryoid bodies forpresence of three germ layer markers TUJI, AFP and SMA.

FIGS. 12A-C is a series of digital images of differentiation markerexpression in cells derived from CD47-null embryoid bodies. FIG. 12Ashows ectoderm differentiation marker expression by cells derived fromCD47-null EB-like clusters formed in serum-free medium. Phase contrastimage of EB-like clusters (a) and differentiation of neural precursorcells from EBs (b and high magnification in c). Neuralmicrotubule-associated protein-2 (MAP2) expression in embryoid bodycells (d) and in a differentiated adherent cell (e). Expression of glialfibrillary acidic protein (GFAP, f), neuron-specific beta III tubulin(g), and S100b astrocyte marker (h) on adherent cells grown fromembryoid bodies in neural differentiation medium. FIG. 12B showsendoderm differentiation marker expression by cells derived fromCD47-null EB-like clusters formed in serum-free medium. Morphology of WTmouse lung endothelial cells in Hepatocyte medium (a), embryoid bodyformation by CD47-null lung endothelial cells in Hepatocyte medium (b),expression of endodermal marker AFP in CD47-null lung endothelial cellsin Hepatocyte medium (c), no expression of AFP in CD47-null endothelialcells grown in EGM2 medium (d), WT mouse lung endothelial cells inmesenchymal medium (e), and CD47 null cells in mesenchymal medium withembryoid body formation (f). Adherent cell outgrowth fromdifferentiating embryoid bodies (g) and differentiated cells stained foradipocyte marker Oil red O staining (h-i). FIG. 12C shows expression ofthe mesoderm marker smooth muscle actin by CD47 null cells grown fromserum-free embryoid bodies transferred into smooth muscledifferentiation medium.

FIGS. 13A-L shows hematopoietic differentiation from CD47-nullendothelial cells. FIGS. 13A-C show representative morphologies ofcolonies generated by growth of CD47-null lung endothelial cells insemisolid medium and FIG. 13D shows a typical rare colony in WTcultures. FIGS. 13E and F show morphology of CD47 null mouse lungendothelial cells in EGM2 medium (FIG. 13E) or L929 conditional medium(FIG. 13F). CD47 null endothelial cells in EGM2 medium do not expressmacrophage marker Mac2 (FIG. 13G), but CD47 null endothelial cells inL929 conditioned medium express Mac2 (FIG. 13H) and show loss of Sca-1expression (FIG. 13I). The cells were confirmed to lack CD47 expression(FIG. 13J). Immunohistochemical detection of Sox2-expression (brown) inrepresentative spleen sections from WT (FIG. 13K) and CD47 null mice(FIG. 13L).

FIGS. 14A-H show additional data for CD47 re-expression effects. FIG.14A shows knockdown of CD47 expression in vivo by CD47-morpholino (MO).FIG. 14B shows re-expression of human CD47-V5 in mouse lung endothelialcells. FIG. 14C shows relative expression of c-MYC (right bars) and CD47(left bars) in transfected cells as compared to that in human umbilicalvein endothelial cells (HUVEC). FIG. 14D shows TSP1 reduces c-MYCexpression in Jurkat cells (left bars for each condition) and when CD47is re-expressed in JinB8 cells (right bars for each condition). FIG. 14Eshows expression level of CD47 in transfected JinB8 cells relative to WTJurkat cells. FIG. 14F-H show CD47 induced cell cytotoxicity in mouselung endothelial cells but not in cells with dysregulated c-Myc: FIG.14F shows re-expression of CD47-FLAG in the presence and absence ofc-Myc-GFP in mouse endothelial cells induced cell cytotoxicity. In eachcondition, from left to right, bars indicate WT, CD47-null, andCD47-null+Myc-GFP. FIG. 14G shows lack of cytotoxicity induced byre-expression of CD47-FLAG in Raji Burkitt's lymphoma cells. FIG. 14Hshows cytotoxicity induced by re-expression of CD47-FLAG in B16 melanomacells, Rat 1 fibroblasts and CD47 null lung endothelial cells. In eachcondition, from left to right, bars indicate B16, rat1 fibroblast, andCD47-null cells.

FIG. 15 is a series of digital images showing projecting neurites fromCD47-null embryoid bodies cultured in neural medium on gelatin coateddishes to induce neuroepithelial differentiation.

FIGS. 16A-C are digital images of human umbilical vein endothelial cells(HUVEC) in continuous culture (FIG. 16A), which become senescent.Treatment with the CD47-binding peptide 7N3 (10 μM; FIG. 16B) or withthe function blocking anti-human CD47 antibody B6H12 (1 μg/ml; FIG. 16C)dramatically increased the sustained proliferation of these cells.

FIG. 17 is a series of digital images of primary WT or TSP1-null murinelung endothelial cells treated with a function blocking anti-mouse CD47antibody (clone 301) or the peptide 7N3. Cells were treated once (1×) ortwice (2×) with Ab301.

FIGS. 18A-C is a series of digital images of HUVEC cells cultured for1-3 weeks after transfection with an antisense CD47 morpholino (FIG.18A). In some cases, the cells were transfected a second time witheither the antisense CD47 morpholino (FIG. 18B) or a mismatch controlmorpholino (mis-mo; FIG. 18C).

FIG. 19 is a series of digital images showing untreated HUVEC cells orHUVEC cells treated with CD47 morpholino which were directly transferredinto neural differentiation medium. Treatment with CD47-morpholinoresulted in sporadic appearance of cells with neuronal phenotypes.

FIGS. 20A and B are a pair of graphs showing proliferation of untreatedHUVEC cells (UT) or HUVEC cells treated with CD47-morpholino (MO1), 7N3peptide, or control peptide 604 assessed using MTS assay. By 6 dayspost-treatment, cells treated with the CD47 binding peptide 7N3 showedenhanced proliferation, whereas control cells treated with the inactivepeptide analog 604 showed decreased proliferation, cells treated withCD47 morpholino showed a slight but not significant enhancement ofproliferation (FIG. 20A; left bars, 72 hours post-treatment; right bars,6 days post-treatment). When the cells were analyzed at 3 weekspost-treatment, cells treated with CD47 morpholino showed significantlyincreased proliferation relative to control HUVEC (FIG. 20B).

FIGS. 21A and B are a pair of graphs showing QPCR analysis of c-Myc mRNAexpression. WT Jurkat T cells were treated with the CD47 binding peptide459 (also known as peptide 4N1) or control peptide 761 at 1 μM or 0.1 μM(FIG. 21A). WT Jurkat T cells were also treated with the CD47-bindingpeptide 7N3 or the control peptide 604 at 1 μm or 10 μM (FIG. 21B).

FIG. 22 is a series of digital images showing direct cardiomyocytedifferentiation of HUVEC following antisense suppression of CD47expression (CD47-MO). The untreated HUVEC were unable to survive in thismedium after 3 days, but the treated cells survived and underwentdifferentiation.

FIG. 23A is a hierarchical cluster analysis of microarray data comparinggene expression of WT and CD47-null endothelial cells, EB-like clustersderived from CD47-null endothelial cells by culture in serum-free mediumfor 36 hours, and V6.5 ES cells. FIG. 23B shows GeneSet EnrichmentAnalysis (GSEA) for ES cell genes as defined by Bhattacharya et al.(Blood 103:2956-2964, 2004) that are induced when CD47 null endothelialcells are induced to form EB-like clusters.

FIG. 24 is a series of graphs showing increased self-renewaltranscription gene levels in kidneys from WT and CD47−/− mice. RT-PCRanalysis of c-Myc (top panel). Klf4 (middle panel), and Oct3/4 (bottompanels) in kidneys from WT and CD47−/− male age matched mice (n=4 ofeach strain).

FIG. 25 is a series of graphs showing that blockade of CD47 elevatesself-renewal transcription factors in human renal cells. Human rTEC weretreated with TSp1 (2.2 nmol/L)±a CD47 monoclonal Ab (clone B6H12, 1μg/ml) and RT-PCR performed for the indicated targets (top, c-Myc;middle, Sox2; bottom, Klf4). Results are the mean (±S.E.M.) of threeseparate experiments.

FIG. 26 is a pair of digital images showing that lack of CD47 signalingprovides for complete generation of a trachea. Orthotopic trachealtransplantation of decellularized tracheal scaffolds, WT-to-CD47-nulland WT-to-WT, was performed. Eight weeks after transplantationdecellularized tracheas in both WT and CD47-null mice displayed basallayer K5+cells (layer below asterisk). However, decellularizedtransplants in CD47-null mice display much more overall cellularrepopulation and complete cartilage restoration (arrows) as compared totransplants in WT.

FIG. 27 is a series of digital images showing that eliminating CD47signaling leads to nephro-genesis in decellularized matrix.Decellularized matrix in WT animals with intact CD47 signaling showsminimal restoration (left panels). The same matrix in animals with CD47signaling blocked display complete restoration with tubular andglomerular like structures and functional vessels (containing red bloodcells) (arrows; right panels).

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. § 1.822. In at least some cases, only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

-   -   SEQ ID NO: 1 is the thrombospondin-1-derived CD47-binding        peptide 7N3 (1102-1112).    -   SEQ ID NO: 2 is the inactive control peptide 604.    -   SEQ ID NOs: 3 and 4 are forward and reverse primers for        detection of murine Nestin expression.    -   SEQ ID NOs: 5 and 6 are forward and reverse primers for        detection of murine Klf4 expression.    -   SEQ ID NOs: 7 and 8 are forward and reverse primers for        detection of murine Sox2 expression.    -   SEQ ID NOs: 9 and 10 are forward and reverse primers for        detection of murine Oct4 expression.    -   SEQ ID NOs: 11 and 12 are forward and reverse primers for        detection of murine Myc expression.    -   SEQ ID NOs: 13 and 14 are forward and reverse primers for        detection of murine E2F expression.    -   SEQ ID NOs: 15 and 16 are forward and reverse primers for        detection of murine p16INK4a expression.    -   SEQ ID NOs: 17 and 18 are forward and reverse primers for        detection of murine TPR53 expression.    -   SEQ ID NOs: 19 and 20 are forward and reverse primers for        detection of murine RB expression.    -   SEQ ID NOs: 21 and 22 are forward and reverse primers for        detection of murine HPRT1 expression.    -   SEQ ID NOs: 23 and 24 are forward and reverse primers for        detection of murine B2M expression.    -   SEQ ID NOs: 25 and 26 are forward and reverse primers for        detection of human B2M expression.    -   SEQ ID NOs: 27 and 28 are forward and reverse primers for        detection of human Myc expression.    -   SEQ ID NOs: 29 and 30 are forward and reverse primers for        detection of human FBP expression.    -   SEQ ID NOs: 31 and 32 are forward and reverse primers for        detection of human HPRT1 expression.    -   SEQ ID NOs: 33 and 34 are forward and reverse primers for        detection of murine TAF9 expression.    -   SEQ ID NO: 35 is an antisense morpholino oligonucleotide        complementary to human and murine CD47.    -   SEQ ID NO: 36 is a 5-base mismatch control morpholino.    -   SEQ ID NO: 37 is a CD47 binding peptide (also known as peptide        459 or 4N1).    -   SEQ ID NO: 38 is the inactive control peptide 761.

DETAILED DESCRIPTION I. Abbreviations

-   -   ANOVA analysis of variance    -   BrdU 5-bromo-2′-deoxyuridine    -   Ca capsule    -   cGMP cyclic guanine monophosphate    -   DMEM Dulbecco's Modified Eagle Medium    -   EB embryoid body    -   EGM endothelial growth medium    -   ES embryonic stem    -   FBS fetal bovine serum    -   GFP green fluorescent protein    -   HUVEC human umbilical vein endothelial cell    -   iPS induced pluripotent stem    -   LIF leukemia inhibitory factor    -   MPSCs multipotent stem cells    -   MTS        3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium    -   NO nitric oxide    -   PBS phosphate buffered saline    -   PSCs pluripotent stem cells    -   RP red pulp    -   sGC soluble guanylyl cyclase    -   TSP1 thrombospondin-1    -   WP white pulp    -   WT wild type

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew el al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers. Inc., 1995 (ISBN 1-56081-569-8).

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term subject includes bothhuman and veterinary subjects, for example, humans, non-human primates,rodents, dogs, cats, horses, and cows.

Administration: Administration of an active compound or composition canbe by any route known to one of ordinary skill in the art.Administration can be local or systemic. Examples of localadministration include, but are not limited to, topical administration,subcutaneous administration, intramuscular administration, intrathecaladministration, intrapericardial administration, intra-ocularadministration, topical ophthalmic administration, or administration tothe nasal mucosa or lungs by inhalational administration. In addition,local administration includes routes of administration typically usedfor systemic administration, for example by directing intravascularadministration to the arterial supply for a particular organ. Thus, inparticular embodiments, local administration includes intra-arterialadministration and intravenous administration when such administrationis targeted to the vasculature supplying a particular organ. Localadministration also includes the incorporation of active compounds andagents into implantable devices or constructs, such as vascular stentsor other reservoirs, which release the active agents and compounds overextended time intervals for sustained treatment effects.

Systemic administration includes any route of administration designed todistribute an active compound or composition widely throughout the body,for example, via the circulatory system. Thus, systemic administrationincludes, but is not limited to intra-arterial and intravenousadministration. Systemic administration also includes, but is notlimited to, topical administration, subcutaneous administration,intramuscular administration, or administration by inhalation, when suchadministration is directed at absorption and distribution throughout thebody by the circulatory system. Systemic administration also includesoral administration, in some examples.

Altered expression: Expression of a biological molecule (for example,mRNA or protein) in a subject or biological sample from a subject thatdeviates from expression if the same biological molecule in a subject orbiological sample from a subject having normal or unalteredcharacteristics for the biological condition associated with themolecule. Normal expression can be found in a control, a standard for apopulation, etc. Altered expression of a biological molecule may beassociated with a disease. The term associated with includes anincreased risk of developing the disease as well as the disease itself.Expression may be altered in such a manner as to be increased ordecreased. The directed alteration in expression of mRNA or protein maybe associated with therapeutic benefits.

Altered protein expression refers to expression of a protein that is insome manner different from expression of the protein in a normal (wildtype) situation. This includes but is not necessarily limited to: (1) amutation in the protein such that one or more of the amino acid residuesis different; (2) a short deletion or addition of one or a few aminoacid residues to the sequence of the protein; (3) a longer deletion oraddition of amino acid residues, such that an entire protein domain orsub-domain is removed or added; (4) expression of an increased amount ofthe protein, compared to a control or standard amount; (5) expression ofan decreased amount of the protein, compared to a control or standardamount; (6) alteration of the subcellular localization or targeting ofthe protein; (7) alteration of the temporally regulated expression ofthe protein (such that the protein is expressed when it normally wouldnot be, or alternatively is not expressed when it normally would be);and (8) alteration of the localized (for example, organ or tissuespecific) expression of the protein (such that the protein is notexpressed where it would normally be expressed or is expressed where itnormally would not be expressed), each compared to a control orstandard.

Controls or standards appropriate for comparison to a sample, for thedetermination of altered expression, include samples believed to expressnormally as well as laboratory values, even though possibly arbitrarilyset, keeping in mind that such values may vary from laboratory tolaboratory. Laboratory standards and values may be set based on a knownor determined population value and may be supplied in the format of agraph or table that permits easy comparison of measured, experimentallydetermined values.

Analog, derivative or mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound. It is acknowledgedthat these terms may overlap in some circumstances.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one light (about 25 kD) and oneheavy chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term antibody includes intact immunoglobulins aswell as a number of well-characterized fragments produced by digestionwith various peptidases, or genetically engineered artificialantibodies. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H) 1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially a Fab withpart of the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,Raven Press, N.Y., 1993). While various antibody fragments are definedin terms of the digestion of an intact antibody, it will be appreciatedthat Fab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies.

Antibodies for use in the methods, compositions, and systems of thisdisclosure can be monoclonal or polyclonal. Merely by way of example,monoclonal antibodies can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495497, 1975)or derivative methods thereof. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL. New York, 1988).

The terms bind specifically and specific binding refer to the ability ofa specific binding agent (such as, an antibody) to bind to a targetmolecular species in preference to binding to other molecular specieswith which the specific binding agent and target molecular species areadmixed. A specific binding agent is said specifically to recognize atarget molecular species when it can bind specifically to that target.

A single-chain antibody (scFv) is a genetically engineered moleculecontaining the V_(H) and V_(L) domains of one or more antibody(ies)linked by a suitable poly peptide linker as a genetically fused singlechain molecule (see, for example. Bird et al., Science, 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988).Diabodies are bivalent, bispecific antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,for example, Holliger et al., Proc. Natl. Acad Sci., 90:6444-6448, 1993;Poljak et al., Structure, 2:1121-1123, 1994). One or more CDRs may beincorporated into a molecule either covalently or noncovalently to makethe resultant molecule an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest. A chimericantibody is an antibody that contains one or more regions from oneantibody and one or more regions from one or more other antibodies.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites.

A neutralizing antibody or an inhibitory antibody is an antibody thatinhibits at least one activity of a target—usually a polypeptide—such asby blocking the binding of the polypeptide to a ligand to which itnormally binds, or by disrupting or otherwise interfering with aprotein-protein interaction of the polypeptide with a secondpolypeptide. An activating antibody is an antibody that increases anactivity of a polypeptide. Antibodies may function as mimics of a targetprotein activity, or as blockers of the target protein activity, withtherapeutic effect derived therein.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse complement), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or plus strand DNA. Sensemolecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules complimentary to adsDNA target. In one embodiment, an antisense molecule specificallyhybridizes to a target mRNA and inhibits transcription of the targetmRNA.

Cell Culture: Cell culture or culturing cells refers to placing cells ina dish, flask, or other container with an appropriate medium (such as agrowth medium or differentiation medium) for the type of cells utilized(such as a medium including glucose, essential amino acids, vitamins,trace elements, salts, a buffer to maintain pH, and/or other componentsfor particular applications).

Differentiation: Refers to the process whereby relatively unspecializedcells (such as embryonic stem cells or other stem cells) acquirespecialized structural and/or functional features characteristic ofmature cells. Similarly, “differentiate” refers to this process.Typically, during differentiation, cellular structure alters andtissue-specific proteins appear.

Differentiation Medium: A synthetic set of culture conditions with thenutrients necessary to support the growth or survival of microorganismsor culture cells, and which allows the differentiation ofundifferentiated cells (such as committed mesenchymal cells) intodifferentiated cells, such as islet cells. Differentiation mediagenerally include glucose, essential amino acids, vitamins, traceelements, salts, a buffer to maintain pH, and/or other components forparticular applications. In one embodiment, a growth medium contains aminimal essential media, supplemented with specific growth factors.

Effective amount of a compound: A quantity of compound sufficient toachieve a desired effect in a subject being treated. An effective amountof a compound can be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, the effectiveamount of the compound will be dependent on the compound applied, thesubject being treated, the severity and type of the affliction, and themanner of administration of the compound.

Expand: A process by which the number or amount of cells in a cellculture is increased due to cell division. Similarly, the terms“expansion” or “expanded” refers to this process. The terms“proliferate,” “proliferation” or “proliferated” may be usedinterchangeably with the words “expand,” “expansion”, or “expanded.”Typically, during an expansion phase, the cells do not differentiate toform mature cells, but divide to form more cells.

Functionally equivalent sequence variant: Sequence alterations thatyield the same results as described herein. Such sequence alterationscan include, but are not limited to, deletions, base modifications,mutations, labeling, and insertions.

Gene expression: The process by which the coded information of a nucleicacid transcriptional unit (including, for example, genomic DNA or cDNA)is converted into an operational, non-operational, or structural part ofa cell, often including the synthesis of a protein. Gene expression canbe influenced by external signals; for instance, exposure of a subjectto an agent that inhibits gene expression. Expression of a gene also maybe regulated anywhere in the pathway from DNA to RNA to protein.Regulation of gene expression occurs, for instance, through controlsacting on transcription, translation. RNA transport and processing,degradation of intermediary molecules such as mRNA, or throughactivation, inactivation, compartmentalization or degradation ofspecific protein molecules after they have been made, or by combinationsthereof. Gene expression may be measured at the RNA level or the proteinlevel and by any method known in the art, including Northern blot,RT-PCR. Western blot, or in vitro, in situ, or in vivo protein activityassay(s).

The expression of a nucleic acid may be modulated compared to a controlstate, such as at a control time (for example, prior to administrationof a substance or agent that affects regulation of the nucleic acidunder observation) or in a control cell or subject, or as compared toanother nucleic acid. Such modulation includes but is not necessarilylimited to overexpression, underexpression, or suppression ofexpression. In addition, it is understood that modulation of nucleicacid expression may be associated with, and in fact may result in, amodulation in the expression of an encoded protein or even a proteinthat is not encoded by that nucleic acid.

Interfering with or inhibiting gene expression refers to the ability ofan agent to measurably reduce the expression of a target gene.Expression of a target gene may be measured by any method known to thoseof ordinary skill in the art, including for example measuring mRNA orprotein levels. It is understood that interfering with or inhibitinggene expression is relative, and does not require absolute suppressionof the gene. Thus, in certain embodiments, interfering with orinhibiting gene expression of a target gene requires that, followingapplication of an agent, the gene is expressed at least 5% less thanprior to application, at least 10% less, at least 15% less, at least 20%less, at least 25% less, or even more reduced. Thus, in some particularembodiments, application of an agent reduces expression of the targetgene by about 30%, about 40%, about 50%, about 60%, or more. In specificexamples, where the agent is particularly effective, expression isreduced by 70%, 80%, 85%, 90%, 95%, or even more. Gene expression issubstantially eliminated when expression of the gene is reduced by 90%,95%, 98%, 99% or even 100%.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. Growth factors include molecules that function asgrowth stimulators (mitogens), factors that stimulate cell migration,factors that function as chemotactic agents or inhibit cell migration orinvasion of tumor cells, factors that modulate differentiated functionsof cells, factors involved in apoptosis, or factors that promotesurvival of cells without influencing growth and differentiation.Examples of growth factors are a fibroblast growth factor (such asFGF-2), epidermal growth factor (EGF), cilliary neurotrophic factor(CNTF), nerve growth factor (NGF), activin-A, and insulin.

Growth medium or expansion medium: A synthetic set of culture conditionswith the nutrients necessary to support the growth (cellproliferation/expansion) of a specific population of cells. In oneembodiment, the cells are stem cells, such as induced pluripotent ormultipotent stem cells. In other examples, the cells are primary cellsobtained from an animal or subject. Growth media generally includeglucose, essential amino acids, vitamins, trace elements, salts, abuffer to maintain pH, and/or other components for particularapplications. In one embodiment, ES growth medium contains a minimalessential media, such as DMEM, supplemented with various nutrients toenhance ES cell growth. Additionally, the minimal essential media may besupplemented with additives such as horse, calf or fetal bovine serum.

Immortalized: Capable of undergoing at least 25, 50, 75, 90, or 95% morecell divisions than a naturally-occurring control cell of the same celltype, genus, and species as the immortalized cell or than the donor cellfrom which the immortalized cell was derived. Preferably, animmortalized cell is capable of undergoing at least 2, 5, 10, or 20-foldmore cell divisions than the control cell. In one embodiment, theimmortalized cell is capable of undergoing an unlimited number of celldivisions. Examples of immortalized cells include cells that naturallyacquire a mutation in vivo or in vitro that alters their normalgrowth-regulating process. Other immortalized cells include cells thathave been genetically modified to express an oncogene, such as ras, myc,abl, bcl2, or neu, or that have been infected with a transforming DNA orRNA virus, such as Epstein Barr virus or SV40 virus (Kumar et al.,Immunol. Lett. 65:153 159, 1999; Knight et al., Proc. Nat. Acad. Sci.USA 85:3130 3134, 1988; Shammah el al., J. Immunol. Methods 160 19 25,1993; Gustafsson and Hinkula, Hum. Antibodies Hybridomas 5:98 104, 1994;Kataoka et al., Differentiation 62:201 211, 1997; Chatelut et al., ScandJ. Immunol. 48:659 666, 1998). Cells can also be genetically modified toexpress the telomerase gene (Roques et al., Cancer Res. 61:8405 8507,2001). In other examples, cells are treated with a substance that makesthem capable of undergoing increased numbers of cell divisions than anuntreated cell of the same type.

Inhibiting protein activity: To decrease, limit, or block an action,function or expression of a protein. The phrase “inhibit proteinactivity” is not intended to be an absolute term. Instead, the phrase isintended to convey a wide range of inhibitory effects that variousagents may have on the normal (for example, uninhibited or control)protein activity. Inhibition of protein activity may, but need not,result in an increase in the level or activity of an indicator of theprotein's activity. By way of example, this can happen when the proteinof interest is acting as an inhibitor or suppressor of a downstreamindicator. Thus, protein activity may be inhibited when the level oractivity of any direct or indirect indicator of the protein's activityis changed (for example, increased or decreased) by at least 10%, atleast 20%, at least 30%, at least 50%, at least 80%, at least 100%₆ orat least 250% or more as compared to control measurements of the sameindicator.

Inhibition of protein activity may also be effected, for example, byinhibiting expression of the gene encoding the protein or by decreasingthe half-life of the mRNA encoding the protein.

Isolated: An isolated biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, for instance,other chromosomal and extrachromosomal DNA and RNA, and proteins.Nucleic acids, peptides and proteins that have been isolated thusinclude nucleic acids and proteins purified by standard purificationmethods. The term also embraces nucleic acids, peptides and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids. The terms isolated and purified do notrequire absolute purity; rather, it is intended as a relative term.Thus, for example, an isolated peptide preparation is one in which thepeptide or protein is more enriched than the peptide or protein is inits natural environment within a cell. Preferably, a preparation ispurified such that the protein or peptide represents at least 50% of thetotal peptide or protein content of the preparation.

Modulator: An agent that increases or decreases (modulates) the activityof a protein or other bio-active compound, as measured by the change inan experimental biological parameter. A modulator can be essentially anycompound or mixture (for example, two or more proteins), such as a NOdonor, a polypeptide, a hormone, a nucleic acid, a sugar, a lipid andthe like.

Morpholino: A morpholino oligo is structurally different from naturalnucleic acids, with morpholino rings replacing the ribose or deoxyribosesugar moieties and non-ionic phosphorodiamidate linkages replacing theanionic phosphates of DNA and RNA. Each morpholino ring suitablypositions one of the standard bases (A, G, C, T/U), so that a 25-basemorpholino oligo strongly and specifically binds to its complementary25-base target site in a strand of RNA via Watson-Crick pairing. Becausethe backbone of the morpholino oligo is not recognized by cellularenzymes of signaling proteins, it is stable to nucleases and does nottrigger an innate immune response through the toll-like receptors. Thisavoids loss of oligo, inflammation or interferon induction. Morpholinoscan be delivered by a number of techniques, including direct injectionto tissues or via infusion pump and intravenous bolus. A morpholino isone example of a stabilized nucleic acid molecule.

Non-immortalized: A cell that cannot divide indefinitely in vitro. Insome embodiments, the non-immortalized cell does not have a nucleic acidmutation that alters its normal growth-regulating process. In someembodiments, the non-immortalized cell does not have two copies of thesame recessive oncogene. In some embodiments, the non-immortalized cellcannot undergo 4-fold, 3-fold, 2-fold, or 1.5-fold more cell divisionsin vitro and retain the same phenotype as the initial cell.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and antisense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers thereof. A nucleotide refers to aribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. A nucleic acid molecule as used herein is synonymous withnucleic acid and polynucleotide. A nucleic acid molecule is usually atleast 10 bases in length, unless otherwise specified. The term includessingle- and double-stranded forms. A polynucleotide may include eitheror both naturally occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of ordinary skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications, such as uncharged linkages (for example,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), charged linkages (for example, phosphorothioates,phosphorodithioates, etc.), pendent moieties (for example,polypeptides), intercalators (for example, acridine, psoralen, etc.),chelators, alkylators, and modified linkages (for example, alphaanomeric nucleic acids, etc.). The term nucleic acid molecule alsoincludes any topological conformation, including single-stranded,double-stranded, partially duplexed, triplexed, hairpinned, circular andpadlocked conformations. Also included are synthetic molecules thatmimic polynucleotides in their ability to bind to a designated sequencevia hydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

Unless specified otherwise, the left hand end of a polynucleotidesequence written in the sense orientation is the 5′ end and the righthand end of the sequence is the 3′ end. In addition, the left handdirection of a polynucleotide sequence written in the sense orientationis referred to as the 5′ direction, while the right hand direction ofthe polynucleotide sequence is referred to as the 3′ direction. Further,unless otherwise indicated, each nucleotide sequence is set forth hereinas a sequence of deoxyribonucleotides. It is intended, however, that thegiven sequence be interpreted as would be appropriate to thepolynucleotide composition: for example, if the isolated nucleic acid iscomposed of RNA, the given sequence intends ribonucleotides, withuridine substituted for thymidine.

An antisense nucleic acid is a nucleic acid (such as, an RNA or DNAoligonucleotide) that has a sequence complementary to a second nucleicacid molecule (for example, an mRNA molecule). An antisense nucleic acidwill specifically bind with high affinity to the second nucleic acidsequence. If the second nucleic acid sequence is an mRNA molecule, forexample, the specific binding of an antisense nucleic acid to the mRNAmolecule can prevent or reduce translation of the mRNA into the encodedprotein or decrease the half-life of the mRNA, and thereby inhibit theexpression of the encoded protein.

Oligonucleotide: A plurality of joined nucleotides joined by nativephosphodiester bonds, between about 6 and about 300 nucleotides inlength. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includestabilized oligonucleotides, such as peptide nucleic acid (PNA)molecules and morpholinos.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington: TheScience and Practice of Pharmacy, The University of the Sciences inPhiladelphia, Editor. Lippincott. Williams, & Wilkins. Philadelphia, PA,21^(st) Edition (2005), describes compositions and formulations suitablefor pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. Incubating includes exposing atarget to an agent for a sufficient period of time for the agent tointeract with a cell. Contacting includes incubating an agent in solidor in liquid form with a cell.

Pluripotent refers to a cell's potential to differentiate into cells ofthe three germ layers: endoderm (e.g., interior stomach lining,gastrointestinal tract, the lungs), mesoderm (e.g., muscle, bone, blood,urogenital), or ectoderm (e.g., epidermal tissues and nervous system).Pluripotent stem cells can give rise to any fetal or adult cell type.Alone they cannot develop into a fetal or adult animal because they lackthe potential to contribute to extra-embryonic tissue (e.g., placenta invivo or trophoblast in vitro).

Pluripotent stem cells (PSCs) are the source of multipotent stem cells(MPSCs) through spontaneous differentiation or as a result of exposureto differentiation induction conditions in vitro. The term multipotentrefers to a cell's potential to differentiate and give rise to a limitednumber of related, different cell types. These cells are characterizedby their multi-lineage potential and the ability for self-renewal. Invivo, the pool of multipotent stem cells replenishes the population ofmature functionally active cells in the body. Among the exemplarymultipotent stem cell types are hematopoietic, mesenchymal, or neuronalstem cells.

Transplantable cells include multipotent stem cells and more specializedcell types such as committed progenitors as well as cells further alongthe differentiation and/or maturation pathway that are partly or fullymatured or differentiated. Exemplary transplantable cells includepancreatic, epithelial, cardiac, endothelial, liver, endocrine, and thelike.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers usually being preferred. The term polypeptideor protein as used herein encompasses any amino acid sequence andincludes modified sequences such as glycoproteins. The term polypeptideis specifically intended to cover naturally occurring proteins, as wellas those that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide thatexhibits at least one useful epitope. The phrase “functional fragment(s)of a polypeptide” refers to all fragments of a polypeptide that retainan activity, or a measurable portion of an activity, of the polypeptidefrom which the fragment is derived. Fragments, for example, can vary insize from a polypeptide fragment as small as an epitope capable ofbinding an antibody molecule to a large polypeptide capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An epitope is a region of a polypeptide capable of binding an immunoglobulin generated in response tocontact with an antigen.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

In some circumstances, variations in the cDNA sequence that result inamino acid changes, whether conservative or not, are minimized in orderto preserve the functional and immunologic identity of the encodedprotein. The immunologic identity of the protein may be assessed bydetermining whether it is recognized by an antibody; a variant that isrecognized by such an antibody is immunologically conserved. Any cDNAsequence variant will preferably introduce no more than twenty, andpreferably fewer than ten amino acid substitutions into the encodedpolypeptide. Variant amino acid sequences may, for example, be 80%, 90%,or even 95% or 98% identical to the native amino acid sequence. Programsand algorithms for determining percentage identity can be found at theNCBI website.

Preventing or treating a disease: Preventing a disease refers toinhibiting the full development of a disease, for example inhibiting thedevelopment of myocardial infarction in a person who has coronary arterydisease or inhibiting the progression or metastasis of a tumor in asubject with a neoplasm. Treatment refers to a therapeutic interventionthat ameliorates at least one sign or symptom of a disease orpathological condition, or interferes with a pathophysiological processafter it has begun to develop. Treatment includes inhibiting orpreventing the partial or full development or progression of a disease,for example in a person who is known to have a predisposition to adisease.

Primary cells: Cells directly obtained or isolated from tissue. Primarycells are not transformed and are not immortalized. These cellsgenerally do not proliferate indefinitely when placed in cell cultureunless they undergo spontaneous immortalization or malignanttransformation. Primary cells obtained from a tissue may include apopulation of multiple cell types, including multiple types ofdifferentiated cells, lineage-committed cells, and/or stem cells (suchas adult stem cells, for example hematopoietic stem cells, mesenchymalstem cells, or neural stem cells). Primary cells obtained from a tissuemay also include primarily a single cell type (or a single cell type maybe isolated or selected from a population of primary cells), such ashuman umbilical vein endothelial cells (HUVEC).

Purified: In a more pure form than is found in nature. The term purifieddoes not require absolute purity; rather, it is intended as a relativeterm. Thus, for example, a purified protein preparation is one in whichthe protein referred to is more pure than the protein in its naturalenvironment within a cell.

The term substantially purified as used herein refers to a molecule (forexample, a nucleic acid, polypeptide, oligonucleotide, etc.) that issubstantially free of other proteins, lipids, carbohydrates, or othermaterials with which it is naturally associated. In one embodiment, asubstantially purified molecule is a polypeptide that is at least 50%free of other proteins, lipids, carbohydrates, or other materials withwhich it is naturally associated. In another embodiment, the polypeptideis at least at least 80% free of other proteins, lipids, carbohydrates,or other materials with which it is naturally associated. In yet otherembodiments, the polypeptide is at least 90% or at least 95% free ofother proteins, lipids, carbohydrates, or other materials with which itis naturally associated.

RNA interference (RNA silencing; RNAi): A gene-silencing mechanismwhereby specific double-stranded RNA (dsRNA) trigger the degradation ofhomologous mRNA (also called target RNA). Double-stranded RNA isprocessed into small interfering RNAs (siRNA), which serve as a guidefor cleavage of the homologous mRNA in the RNA-induced silencing complex(RISC). The remnants of the target RNA may then also act as siRNA; thusresulting in a cascade effect.

Senescence: The biological process(es) of aging and showing the effectsof increased age. In one embodiment, a senescent cell does not divideand/or has a reduced capacity to divide.

Small molecule inhibitor: A molecule, typically with a molecular weightless than 1000, or in some embodiments, less than about 500 Daltons,wherein the molecule is capable of inhibiting, to some measurableextent, an activity of some target molecule.

Stabilized nucleic acid molecules: A variety of synthetic nucleic acidderivatives with increased stability as compared to native (e.g.,non-modified) nucleic acids. Stabilized nucleic acid molecules includenucleic acids where the labile phosphodiester bonds in nucleic acids arereplaced with more stable phosphoramidates or peptide amide backbones,or oligonucleotides including one or more such nucleic acid derivatives.Also included are nucleic acids having a substitution of thedeoxyribosyl moiety with a more stable morpholine derivative (e.g.,morpholinos) or oligonucleotides including one or more morpholinonucleic acids. In other examples, stabilized nucleic acid moleculesinclude “locked” nucleic acids where the ribose moiety is modified witha bridge connecting the 2′ oxygen and the 4′ carbon, or oligonucleotidesincluding one or more locked nucleic acid.

Stem cell: A cell that can generate a fully differentiated functionalcell of a more than one given cell type. The role of stem cells in vivois to replace cells that are destroyed during the normal life of ananimal. Generally, stem cells can divide asymmetrically without limitand may be lineage-committed, totipotent, or pluripotent. Afterdivision, the stem cell may remain as a stem cell, become a precursorcell, or proceed to terminal differentiation. A nervous system stem cellis, for example, a cell of the central nervous system that canself-renew and can generate astrocytes, neurons and oligodendrocytes.

A “somatic precursor cell” is a cell that can generate a fullydifferentiated functional cell of at least one given cell type from thebody of an animal, such as a human. A neuronal precursor cell cangenerate of fully differentiated neuronal cell, such as, but not limitedto, and adrenergic or a cholinergic neuron. A glial precursor cell cangenerate fully differentiated glial cells, such as but not limited toastrocytes, microglia and oligodendroglia. Generally, precursor cellscan divide and are pluripotent. After division, a precursor cell canremain a precursor cell, or may proceed to terminal differentiation. Aneuronal precursor cell can give rise to one or more types of neurons,such as dopaminergic, adrenergic, or serotonergic cells, but is morelimited in its ability to differentiate than a stem cell. In oneexample, a neuronal stem cell gives rise to all of the types of neuronalcells (such as dopaminergic, adrenergic, and serotonergic neurons) butdoes not give rise to other cells, such as glial cells.

Target sequence: A target sequence is a portion of ssDNA, dsDNA, or RNAthat, upon hybridization to a therapeutically effective oligonucleotideor oligonucleotide analog (e.g., a morpholino), results in theinhibition of expression of the target. Either an antisense or a sensemolecule can be used to target a portion of dsDNA, as both willinterfere with the expression of that portion of the dsDNA. Theantisense molecule can bind to the plus strand, and the sense moleculecan bind to the minus strand. Thus, target sequences can be ssDNA,dsDNA, and RNA.

Totipotent or totipotency refers to a cell's ability to divide andultimately produce an organism and its extra-embryonic tissues in vivo.In one aspect, the term “totipotent” refers to the ability of the cellto progress through a series of divisions into a blastocyst in vitro.The blastocyst comprises an inner cellular mass (ICM) and a trophoblast.By ICM is meant the cells surrounded by the trophectoderm. The innercell mass cells give rise to most of the fetal tissues upon furtherdevelopment. The cells found in the ICM give rise to pluripotent stemcells that possess the ability to proliferate indefinitely, or ifproperly induced, to differentiate into all cell types contributing toan organism. By “trophectoderm” is meant the outermost layer of cellssurrounding the blastocoel during the blastocyst stage of primateembryonic development. Trophectoderm becomes trophoblast and gives riseto most or all of the placental tissue upon further development.Trophoblast cells generate extra-embryonic tissues, including placentaand amnion.

Therapeutic: A generic term that includes both diagnosis and treatment.

Therapeutically effective amount: A quantity of compound sufficient toachieve a desired effect in a subject being treated. An effective amountof a compound may be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, the effectiveamount will be dependent on the compound applied, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration of the compound. For example, a therapeutically effectiveamount of an active ingredient can be measured as the concentration(moles per liter or molar-M) of the active ingredient (such as a smallmolecule, peptide, protein, oligonucleotide, or antibody) in blood (invivo) or a buffer (in vitro) that produces an effect.

Tissue Matrix: A scaffold having a three-dimensional structure of anorgan, tissue, or portion thereof, but substantially lacking cellularcontent. A tissue matrix can be a decellularized organ (for example,liver, kidney, heart, lung, bladder, trachea, or esophagus) or portionthereof or tissue (for example, vessel, valve, skin, bone, joint,airway, urethra, nerve, cornea, retina, inner ear, muscle, orcartilage). The decellularized organ or tissue preserves the compositionand structure of the extracellular matrix of the organ but the cells aresubstantially removed. A tissue matrix also includes a synthetic organ(or portion thereof) or tissue scaffold made of synthetic biocompatibleextracellular matrices that can support tissue regeneration.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, includesadministering a therapeutically effective amount of a composition thatincludes a peptide, antibody, or oligonucleotide (e.g., morpholino),sufficient to enable the desired activity.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word or is intendedto include and unless the context clearly indicates otherwise. Hence“comprising A or B” means “including A. or including B, or including Aand B.” It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

In case of conflict, the present specification, including explanationsof terms, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

III. Thrombospondin and CD47

Thrombospondin 1 (TSP1; also known as THBS1) is an extracellularsecreted protein that is involved in a myriad of cellular processes,including platelet aggregation, neurite outgrowth, cell motility, cellsurvival, and cellular proliferation. Among TSP1's best-characterizedfunctions is inhibition of angiogenesis. Angiogenesis ameliorates thepoor oxygenation of damaged tissue that is a limiting factor for patientrecovery in a variety of clinical settings, including surgery, burnwound healing, amputation, stroke, pulmonary arterial hypertension,peripheral vascular disease, and myocardial infarction. Because it isdesirable to promote angiogenesis within these contexts, antagonizingTSP1's activity has been a valuable research objective. Additionally,tumors require vascularization for growth. Agents that mimic the abilityof TSP1 to inhibit angiogenesis are therefore considered possibletherapies for cancer. In vitro studies have shown the ability of suchagents to block tumor driven angiogenesis. In vivo results in animalshave also been encouraging and have led to clinical trials in people.See Rusk et al., Clin Cancer Res 12:7456-7464, 2006; Markovic et al., AmJ Clin Oncol 30:303-30), 2007.

TSP1 contains three type 1 repeat structural domains and acarboxy-terminal domain that were identified as the loci of thefull-length protein's anti-angiogenic functionality (Lawler, Curr. Opin.Cell Biol. 12(5): 634-640, 2000). TSP1 sequences are publicallyavailable, such as GenBank Accession Nos. NM_003246 and NM_011580(nucleic acids) and NP_003237 and NP_035710 (protein), all of which areincorporated herein by reference as present in GenBank on Dec. 10, 2012.One of ordinary skill in the art can identify additional TSP1 sequences,including variant sequences.

Overexpression of TSP1 has been observed in ischemic tissue, and isproposed to regulate angiogenesis within ischemic tissue (Favier et al.,J Pathol. 207(3): 358-366, 2005), since TSP1 preferentially interfereswith wound healing-associated angiogenesis (Streit et al., EMBO J.19(13): 3272-3282, 2000) and limits revascularization in a model of hindlimb ischemia similar to that employed by the current inventors (Kopp etal., J. Clin. Invest. 116(12): 3277-3291, 2006). Peptides derived fromthe type 1 repeats inhibit angiogenesis (Shafiee et al., IOVS 41(8):2378-2388, 2000; Yee et al., Am J. Pathol. 165(2): 541-552, 2004; Tolsmaet al., J. Cell Biol. 122: 497-511, 1993; Armstrong and Bornstein. Mat.Biol. 22(1): 63-71, 2003; Guo et al., Cancer Res. 58(14): 3154-3162,1998; Guo et al., J. Peptide Res 50:210-221, 1997). Additional TSP1peptides (e.g., 4N1 and 7N3 classes) have previously been described;see, e.g., U.S. Pat. Nos. 5,399,667; 5,627,265; 6,469,138; 5,357,041;5,491,130; 5,770,563; 5,849,701; 6,051,549; 6,384,189; 6,458,767; and7,129,052.

TSP1 acts through several cellular receptors, including CD36 andintegrin-associated protein (IAP)/CD47. It was originally thought thatTSP1 exerted its anti-angiogenic effects by acting through CD36 (Quesadaet al., Cell Death and Diff, 12:649-658, 2005; Jimenez et al., Nat Med.6(1):41-48, 2000: de Fraipon et al., Trends Mol. Med. 7(9):401-407,2001). However, CD36 is unlikely to be responsible for theanti-angiogenic actions of TSP1. For example, short peptides comprisedof the TSP1 type 1 repeat can inhibit FGF- and VEGF-induced migration ofhuman endothelial cells that lack CD36 binding (Vogel et al., J. Cell.Biochem. 53:74-84, 1993, Guo et al., J. Peptide Res 50:210-221, 1997;Short et al., J Cell Biol. 168(4): 643-653, 2005). A sequence in thecarboxy-terminal domain of TSP1 that binds to CD47 inhibits nitricoxide-mediated pro-angiogenic signaling (Isenberg et al., J. Biol. Chem.281:26069-26080, 2006) and was shown to have anti-angiogenic activity(Kanda et al., Exp Cell Res. 252(2):262-72, 1999). RecombinantC-terminal domain of TSP1 that contains this sequence and binds to CD47also inhibits NO signaling in endothelial cells and was shown to haveanti-angiogenic activity (Kanda et al., Exp Cell Res. 252(2):262-72,1999) in CD36-null, but not CD47-null cells. In contrast with theresults from TSP1-derived peptides, the use of oligonucleotides toinhibit production of TSP1 suggested a contributory role of TSP1 inexcisional dermal wound healing (DiPietro et al., Am J. Pathol. 148(6):1851-1860, 1996). This activity is mediated by regulation of thechemokine MIP1. In contrast, ischemic wounds heal better in mice lackingeither TSP1 or CD47 and display more vigorous angiogenic responses(Isenberg et al., Ann. Surg. 247:860-868, 2008). CD36 null mice showedno advantage for healing ischemic wounds, revealing that theanti-angiogenic activity of TSP1 in an ischemic environment is mediatedby CD47 rather than CD36. Likewise, in skin graft healing enhanced grafttake is obtained in CD47 null wounds compared to either WT or CD36 nullwounds.

CD47 is an atypical member of the immunoglobulin and the Gprotein-coupled receptor superfamilies. It consists of an N-terminalextracellular IgV set domain, 5 transmembrane segments and analternatively spliced cytoplasmic tail (Brown and Frazier, Trends CellBiol. 11(3): 130-135, 2001). CD47 sequences are publically available,such as GenBank Accession Nos. NM_198793, NM_001777, and NM_010581(nucleic acids) and NP_942088. NP_001768, and NP_034711 (protein), allof which are incorporated herein by reference as present in GenBank onDec. 10, 2012. One of ordinary skill in the art can identify additionalCD47 sequences, including variant sequences.

Although identified earlier as “integrin associated protein” (IAP), CD47was discovered to be a high affinity receptor for the C-terminal domainof TSP1 in 1996 (Gao et al., J. Biol. Chem. 271: 21-24, 1996. Isenberget al., J. Biol. Chem. 284: 1116-1125, 200)). Two members of the signalinhibitory receptor protein family, SIRPα (also known as BIT, SHPS-1 andp84) and SIRPγ are cell-bound counter receptors for CD47 (van Beek etal., J. Immunol. 175:7781-87, 2005). CD47 is expressed on many if notall normal cells, and signals in part through coupling to heterotrimericG proteins of the G_(i) class (Frazier et al., J. Biol Chem.274:8554-8560, 1999).

TSP1, via binding to CD47, potently limits physiologic NO signaling inall vascular cell types including endothelial cells, vascular smoothmuscle cells, and platelets and inflammatory cells. TSP1-CD47 signalingalso directly and acutely regulates tissue blood flow and arterial toneby inhibiting NO-driven vasorelaxation, and exerts anti-vasorelaxiveeffects on smooth muscle by antagonizing the ability of NO to stimulatecGMP synthesis (Isenberg et al., Proc Natl Acad Sci USA. 102(37):13141-13146, 2005; Isenberg et al., Cardiovasc Res., 71(4):785-793,2006); Isenberg et al., J Biol Chem 281:26069-26080, 2006, Isenberg etal., Blood, 109(5):1945-1952, 2007) and through its ability to rapidlyupregulate NADPH-oxidase (Nox) to increase production of superoxide, apotent NO scavenger (Csanyi et al., Artherioscl. Thromb. Vasc. Biol.32:2966-73, 2012). Though inhibition of NO signaling may be induced byTSP1 interacting with CD36, this effect occurs at doses 100- to1000-fold greater than the doses of TSP1 that drive inhibition throughCD47. Also, TSP1 inhibition of NO signaling through CD36 cannot occur inthe absence of CD47 at any dose; thus, the physiologically relevantpathway is via CD47 (Isenberg et al., J Biol Chem. 281(36):26069-26080,2006). See also International Patent Publication No. WO 2008/060785,which is incorporated herein by reference in its entirety.

The structure and function of CD47 has been explored using anti-CD47antibodies and peptide ligands of the receptor. Certain anti-CD47 andTSP1-derived CD47 ligands initiate cell death in breast cancer celllines (Manna and Frazier, Cancer Res. 64:1026-1036, 2004) and Jurkat Tcells (Manna and Frazier, J Immunol. 170(7):3544-3553, 2003). These. andsimilar experiments, led to the hypothesis that CD47 is necessary forFAS-mediated apoptosis of Jurkat T cells (Manna et al., J Biol. Chem.280(33):29637-29644, 2005). Synthetic peptides derived from thefull-length sequence of CD47 have been used to probe its structure(Rebres et al., J. Biol. Chem. 276(37):34607-34616, 2001). Ligation ofCD47 induces actin polymerization (Rebres et al., J. Biol. Chem.276(10):7672-7680, 2001), and its ligation by peptides derived from thecarboxy-terminus of TSP1 stimulates the integrin-mediated adhesion ofmelanoma cells to specific substrates (Barazi et al., J. Biol. Chem.277(45):42859-42866, 2002; Gao et al., J. Cell Biol. 135(2):533-544,1996).

Different antibodies engaging CD47 can exert opposing stimulatory andinhibitory effects on cells (Li et al., J Immunol 166:2427-2436, 2001;Waclavicek et al., J Immunol 159:5345-5354, 1997: Pettersen et al., JImmunol 162:7031-7040, 1999; Ticchioni et al., J Immunol 158:677-684,1997). Likewise, a specific CD47 ligand can act as an agonist or anantagonist in different contexts. For instance. CD47 ligation by aparticular ligand may have different effects in isolated cells than invivo. Therefore, some effects of CD47 antibodies that have been definedusing isolated cells do not extrapolate to in vivo activities, and thefunction of a specific CD47 ligand in vivo cannot be predicted solely onthe basis of in vitro testing. However, agents that block CD47 functionin vitro consistently show protective activities in mouse, rat, and pigmodels of stress. These include fixed ischemia, ischemia-reperfusion,and radiation injury (Maxhimer et al., Plast. Reconstr. Surg.124:1880-1889, 2009; Maxhimer et al., Si. Transl. Med. 1:3ra7, 2009).Some of this tissue protection is mediated by increased NO/cGMPsignaling, but additional cytoprotective pathways are also involved,including mitigation of pathologic reactive oxygen species (Bauer etal., Cardiovasc. Res. 88: 471-481, 2010; Csanyi et al., Artherioscl.Thromb. Vasc. Biol. 32:2966-73, 2012). For example, radioprotectioncaused by CD47 blockade involves activation of a protective autophagypathway (Soto-Pantoja et al., Autophagy 8:1628-1642, 2012). Thisprotective autophagy response is evident in isolated cells and intissues of an irradiated mouse. Furthermore, the proliferative andsurvival advantage of cells lacking CD47 or TSP1 described herein revealanother important pro-survival activity of CD47 blockade that isconserved in isolated cells and living tissues of mammals. Without beinglimited by theory, this activity appears to be mediated by overcomingTSP1/CD47 signaling that limits the self-renewal and reprogrammingcapacities of cells via inhibiting the expression of c-Myc and othertranscription factors that are critical for stem cell maintenance.

IV. Generation of Pluripotent and Multipotent Cells and DifferentiatedCells

Disclosed herein are methods for generating or inducing pluripotent ormultipotent stem cells, methods for generating lineage-committed ordifferentiated cells, and methods for maintaining and/or expanding stemcells or differentiated cells in culture. It is shown herein thatblockade of CD47/TSP1 signaling dramatically increases the proliferativecapacity of primary cells and also induces expression of stem cellmarker genes (such as c-Myc, Sox2, Klf4, and Oct4). These cells arecapable of forming embryoid bodies (EBs) or EB-like clusters anddifferentiation into many different cell types upon exposure to suitableculture conditions (such as culture with a differentiation medium).Thus, the disclosed methods include contacting cells (such as primarycells, stem cells, or differentiated cells) with one or more agents thatblock CD47 signaling. Without being bound by theory, it is believed thatin at least some cases, primary cells isolated from an animal containlineage-committed stem cells that can become multi- or pluripotent whenCD47 signaling is blocked.

A. Inducing Pluripotent or Multipotent Stem Cells

In particular embodiments, the described methods include obtainingprimary cells (such as lineage-committed (differentiated) cells) from ananimal or subject, culturing the primary cells, and contacting theobtained cells with an agent that can block CD47 signaling. Multipotentor induced pluripotent stem cells are produced from the CD47-blockedcells when the blocked cells are cultured in appropriate culture media,which in particular embodiments is a serum-free medium.

In some examples, the methods include obtaining primary cells from ananimal (such as a human or a non-human mammal). Primary cells can beobtained from any tissue of interest, including without limitation,liver (e.g., hepatocytes), lung (e.g., lung endothelial cells), bonemarrow (such as myeloid cells or lymphoid cells), spleen, skin (e.g.,fibroblasts, melanocytes, or keratinocytes), adipose tissue (e.g.,adipocytes or mesenchymal cells), heart (e.g., cardiomyocytes or cardiacvalve endothelial cells), smooth muscle, blood vessels (e.g., vascularsmooth muscle or vascular endothelial cells, such as umbilical veinendothelial cells), lymph vessels (e.g., lymphatic endothelial cells),skeletal muscle (e.g., myoblasts), tendons (e.g., tenocytes), neuraltissue (e.g., neurons, astrocytes, or glial cells), bone (e.g.,osteocytes), pancreas (e.g., islet cells), oral or nasal mucosalbiopsies, dental pulp, or hair follicles. In particular examples,primary cells can be obtained from adipose tissue (such as adipocytes),dermal biopsy (such as mesenchymal fibroblasts), or bone marrowaspirates (such as hematopoietic precursors, also referred to ashemangioblasts or hematopoietic stem cells). In additional examples,primary cells can be obtained from umbilical cord or umbilical cordblood or foreskins from newborns (such as fibroblasts, keratinocytes,and/or microvascular endothelial cells).

Primary cells obtained from a tissue may include a population ofmultiple cell types, including multiple types of differentiated cells,lineage-committed cells, and/or stem cells (such as adult stem cells,for example hematopoietic stem cells, mesenchymal stem cells, or neuralstem cells). Primary cells obtained from a tissue may also includeprimarily a single cell type or a single cell type may be isolated orselected from a population of primary cells.

Methods for obtaining primary cells are known to one of ordinary skillin the art. For example, a tissue or a portion thereof is collected froman animal, incubated with an enzyme to release cells (such ascollagenase, trypsin, or pronase) in a growth medium for a period oftime sufficient to dissociate the cells (such as about 5 minutes to 2hours), and plated in a cell culture dish with growth medium. Cells areincubated at a temperature of about 37° C. (such as about 34° C. toabout 39° C.) in an atmosphere containing about 5% CO₂ (such as about4-6% CO₂). Primary cells are also commercially available, for examplefrom Lonza (Basel, Switzerland), Life Technologies (Carlsbad, CA),PromoCell (Heidelberg, Germany), and ScienCell (Carlsbad, CA), and alsofrom the American Type Culture Collection (Manassas, VA) or other cellrepositories.

In some examples, primary cells (such as primary cells obtained from asubject) are placed in a cell culture dish with an appropriate cellculture medium for the type of primary cells utilized (such as a mediumincluding glucose, essential amino acids, vitamins, trace elements,salts, a buffer to maintain pH, and/or other components for particularapplications). For example, if the primary cells are endothelial cells(such as lung endothelial cells or HUVECs), the cell culture medium isan endothelial cell growth medium. In one particular example, theendothelial cell growth medium is EGM2 (Lonza, Basel, Switzerland),which includes hydrocortisone, hEGF, VEGF, hFGFb, R3-IGF-1, fetal bovineserum, ascorbic acid, heparin, and gentamicin/amphotericin B. In otherexamples, if the primary cells are epithelial cells (such ashepatocytes), the cell culture medium is a hepatocyte cell culturemedium and if the primary cells are fibroblasts, the cell culture mediumis a fibroblast cell culture medium. One of ordinary skill in the artcan select an appropriate cell culture medium for a particular type ofprimary cell. Primary cell culture media are also commerciallyavailable, for example from Lonza, Life Technologies (Carlsbad, CA), BDBiosciences (San Jose, CA), and Sigma-Aldrich (St. Louis, MO). In someexamples, the primary cells may be cultured for at least 1 day (such asat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days)prior to contacting the cells with an inhibitor of CD47 signaling. Inother examples, the primary cells are cultured for at least 1 passage(such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 passages) prior tocontacting the cells with an inhibitor of CD47 signaling.

The primary cells (such as cultured primary cells) are contacted with aneffective amount of an inhibitor of CD47 signaling. Inhibitors of CD47signaling are discussed in detail in Section V, below. In some examples,the inhibitor is included in the culture medium (for example, if theinhibitor is a peptide, antibody, or small molecule). In other examples,the cells are transformed or transfected with the inhibitor (forexample, if the inhibitor is an antisense or stabilized oligonucleotide,such as a morpholino oligonucleotide, or a plasmid encoding a siRNA ordsRNA). One of ordinary skill in the art can select an appropriate modefor contacting the cells with the inhibitor.

The cells are contacted with the inhibitor of CD47 signaling for aperiod of time sufficient to achieve the desired effect, such asgeneration or expansion of iPS or multipotent stem cells. In someexamples, presence of iPS or multipotent stem cells in the culture isidentified by increased expression of c-Myc, SSEA1, c-Kit, Sca-1,nestin, Nanog, or other stem cell markers or increased ability of thecells to proliferate in culture (for example as compared to an untreatedcell of the same type). In some examples, the expression of stem cellmarkers (such as c-Myc, Sox2, Klf4, nestin, Nanog, or Oct4) in cellstreated with a CD47 signaling inhibitor is increased by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold or more ascompared to a control. In other examples, the cells treated with a CD47signaling inhibitor proliferate in culture for at least one more passage(such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more passages) or atleast one more day (such as 2, 3, 4, 5, 6, 7, 10 days, 2, 3, 4, 5, 6, 7weeks, or 2, 3, 4, 5, 6 months or more) as compared to a control.

The cells are contacted with an amount of the CD47 signaling inhibitorthat is sufficient to achieve the desired effect, such as generation orexpansion of iPS or multipotent stem cells, as discussed above. In someembodiments, the cells are contacted with a peptide, antibody, or smallmolecule inhibitor, which can be included in the cell culture medium. Insome examples, the inhibitor is a peptide (such as a CD47 bindingpeptide, for example SEQ ID NO: 1 or SEQ ID NO: 37, disclosed herein).The cells are contacted with about 1 nM to 100 mM peptide (such as about10 nM to 10 mM, 100 nM to 1 mM, 100 nM to 10 μM, or 1 μM to 100 μM). Insome examples, the cells are contacted with about 1 μM peptide (forexample, about 1 μM 7N3 peptide). The cells are contacted with thepeptide for at least 1 day and can be contacted with the peptidecontinuously, for any desired period of time for the maintenance and/orexpansion of the cells. In some examples, the cells are contacted withthe peptide for about 1, 2, 3, 4, 5, 6, 7, 10 days, about 2, 3, 4, 5, 6,7 weeks, or about 2, 3, 4, 5, 6 months or more. In additional examples,the cells are contacted with the peptide transiently, for about 1 day to4 weeks or more (such as about 1, 2, 3, 4, 5, 6, 7 days, 2, 3, 4, weeks,or more) and then are subsequently maintained in culture without thepeptide for about 1 week or more. In some embodiments, the cells arecontacted with a peptide which is in solution in the tissue culturemedium.

In other embodiments, the cells are contacted with a peptide which isimmobilized on a tissue culture substrate, a natural tissue matrix, or asynthetic matrix by adsorption or covalent attachment.

In other examples, the cells are contacted with an anti-CD47 antibody,including, but not limited to B6H12 (e.g., Gresham et al., J. Cell Biol.108:1935-1943, 1989, and Brown et al., J. Cell Biol. 111:2785-2794,1990; for example, commercially available from Santa Cruz Biotechnology,as catalog number sc-12730), MIAP301 (e.g., Chang et al., Neuroscience102(2):289-296, 2001; commercially available for instance from RDIDivision of Fitzgerald Industries Intl., as catalog numberRDI-MCD47-301), or OX101 (for example, commercially available from SantaCruz Biotechnology, as catalog number sc-53050). In additional examples,the cells are contacted with an anti-TSP1 antibody, such as A6.1 or C6.7(see, e.g., Annis et al., J. Thromb. Haemost. 4:459-468, 2006: Abcamcatalog numbers ab1823 and ab140257, respectively). The cells arecontacted with about 10 ng/ml to 1 mg/ml antibody (such as about 100ng/ml to 500 μg/ml, 500 ng/ml to 100 μg/ml, or 100 ng/ml to 10 μg/ml, 1μg/ml to 50 μg/ml, or 1 μg/ml to 10 μg/ml). In some examples, the cellsare contacted with about 1 μg/ml of the antibody. The cells arecontacted with the antibody for at least 1 day and can be contacted withthe antibody continuously, for any desired period of time for themaintenance and/or expansion of the cells. In some examples, the cellsare continuously contacted with the antibody for about 1, 2, 3, 4, 5, 6,7, 10 days, about 2, 3, 4, 5, 6, 7 weeks, or about 2, 3, 4, 5, 6 monthsor more. In additional examples, the cells are transiently contactedwith the antibody for example, for about 1 day to 4 weeks or more (suchas about 1, 2, 3, 4, 5, 6, 7 days, 2, 3, 4, weeks, or more) and then aresubsequently maintained in culture without the antibody for about 1 weekor more. In some embodiments, the cells are contacted with an antibodywhich is in solution in the tissue culture medium. In other embodiments,the cells are contacted with an antibody which is immobilized on atissue culture substrate, a natural tissue matrix, or a synthetic matrixby adsorption or covalent attachment.

In additional examples, the inhibitor is a small molecule inhibitor ofCD47 signaling. The cells are contacted with about 0.1 nM to 1 M of thesmall molecule inhibitor (such as about 1 nM to 100 mM, 10 nM to 10 mM,100 nM to 1 mM, 100 nM to 10 μM, or 1 μM to 100 μM). The cells arecontacted with the small molecule for at least 1 day and can becontacted with the small molecule continuously, for any desired periodof time for the maintenance and/or expansion of the cells. In someexamples, the cells are continuously contacted with the small moleculefor about 1, 2, 3, 4, 5, 6, 7, 10 days, about 2, 3, 4, 5, 6, 7 weeks, orabout 2, 3, 4, 5, 6 months or more. In additional examples, the cellsare transiently contacted with the small molecule, for example for about1 day to 4 weeks or more (such as about 1, 2, 3, 4, 5, 6, 7 days, 2, 3,4, weeks, or more) and then are subsequently maintained in culturewithout the small molecule for about 1 week or more. In someembodiments, the cells are contacted with a small molecule which is insolution in the tissue culture medium.

In other embodiments, the cells are contacted with an oligonucleotideinhibitor of CD47 signaling (such as an antisense or stabilizedoligonucleotide complementary to CD47 or TSP1), which can be introducedto the cells by transfection or transformation. The oligonucleotideinhibitor can include without limitation antisense, inhibitory RNA(RNAi), small inhibitory RNA (siRNA), short hairpin RNA (shRNA),microRNA (miRNA), lncRNA, and circRNA oligonucleotides. Methods forintroducing nucleic acids to cells are known to one of ordinary skill inthe art, and include but are not limited to, liposomal-mediatedtransfection, electroporation, and conjugation of the oligonucleotidecompound to a cell-penetrating peptide.

Transfection of oligonucleotides generally involves the use ofliposomal-mediated transfection reagents (such as LIPOFECTAMINE™), anumber of which are commercially available. Methods for transfection andelectroporation of nucleic acids, including antisense compounds, arewell known in the art (see, for example, U.S. Pat. Nos. 7,307,069 and7,288,530; Pretchtel et al., J. Immunol. Methods 311(1-2):139-52, 2006;Ghartey-Tagoe et al., Int. J. Pharm. 315(1-2):122-133, 2006, each ofwhich are herein incorporated by reference). In additional examples, theoligonucleotides can be delivered with a vector, such as a viral vector(for example, an adenovirus, lentivirus, or adeno-associated virusvector). In still further examples, the oligonucleotide can be deliveredto the cells by an endocytosis-mediated process (e.g., ENDO-PORTER, GeneTools, Inc., Corvallis, OR: U.S. Pat. No. 7,084,248). About 1 nM to 100mM oligonucleotide (such as about 10 nM to 10 mM, 100 nM to 1 mM, 0.1 μMto 10 μM, 1 μM to 100 μM, 1 μM to 10 μM or 2.5 μM) is transfected orotherwise introduced to the cells. Introduction of the oligonucleotideto the cells can be repeated one or more times if desired. For example,the cells can be transfected (or otherwise treated) at intervals of 1day or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days,or more). In other examples, a single exposure of cells to theoligonucleotide (even in the absence of transfection techniques oragents) or single dose to a subject is used. In particular examples, theinhibitor is an antisense morpholino oligonucleotide complementary toCD47 (such as SEQ ID NO: 35).

In additional embodiments, the methods include generating embryoidbodies or EB-like clusters from the iPS or multipotent stem cellsgenerated as described above. In some examples, cells which have beencontacted with an agent that inhibits CD47 signaling are transferred toserum-free medium and cultured for at least one day (such as 1, 2, 3, 4,5, 6, 7 days or more). In some examples, embryoid bodies or EB-likeclusters are maintained in culture for at least 1 day or more (such asat least about 1, 2, 3, 4, 5, 6 days; about 1, 2, 3, 4, 5, 6 weeks; orabout 1, 2, 3 or more months). One of ordinary skill in the art canidentify formation of EBs, such as by morphology (for example, formationof cell aggregates) or expression of pluripotent stem cell markers (forexample, alkaline phosphatase, SSEA-1, c-Kit, nestin. Nanog. Oct4, Sox2,and/or Klf4).

B. Generating Differentiated Cells

In some embodiments, the disclosed methods include producing a desireddifferentiated cell type from a previously lineage-committed cell type.Desired cell types can be produced by generating multipotent or iPScells using a CD47 blocking agent as described above, and then culturingthe iPS cells in media containing appropriate differentiating factors.The newly-differentiated cells can also be immortalized for storage.Such cells will maintain their differentiated state in the appropriatemedia, which can be selected by one of ordinary skill in the art.

Induced pluripotent or multipotent stem cells or EBs are produced asdescribed above. Cells are then transferred to a differentiation mediumcontaining factors appropriate for obtaining the desired cell type(s).In some examples, the differentiation medium includes one or more agentsthat inhibit CD47 signaling. In other examples, the differentiationmedium does not include an agent that inhibits CD47 signaling. One ofordinary skill in the art can select appropriate differentiation media,including, but not limited to those described below. In someembodiments, the methods include obtaining primary cells from a subject,culturing the primary cells, contacting the primary cells with an agentthat blocks CD47 signaling, and isolating cells that express at leastone stem cell marker (such as at least 1, 2, 3, 4, 5, or more stem cellmarkers) from the cells contacted with the CD47 inhibitor. In someexample, the stem cell markers include one or more of c-Myc, Oct4, Sox2,Klf4, Nanog, SSEA1, c-Kit, or Sca-1. The cells that express the one ormore stem cell markers are then cultured in serum-free medium to produceiPS or multipotent stem cells and culturing the iPS or multipotent stemcells in a cell differentiation medium to produce differentiated cells.

In some examples, the iPS or multipotent stem cells or EBs are culturedin a differentiation medium that results in generation of cells havingcharacteristics of ectoderm-derived lineages (such as neural cells, forexample, neurons, astrocytes, glia, cranial or sensory neurons and/organglia; pigment cells: head connective tissues, epidermis, mammarygland, or hair). In a particular example, iPS cells are transferred intoa neural differentiation medium, such as serum-free EBM basal medium(for example, commercially available from Lonza, Basel, Switzerland)supplemented with FGF2 and EGF (about 5-20 ng/ml), heparin, andgentamycin sulfate. In some examples, cells form neurospheres (EBs) in1-2 days, which are then plated onto non-tissue culture dishes in thesame medium, but lacking heparin. In other examples, the neurospheresare dispersed and cultured with EBM basal medium supplemented with FGF2and EGF (about 5-20 ng/ml), gentamycin sulfate, and StemPro Neuralsupplement (Life Technologies, Carlsbad, CA). In some examples, themedium includes one or more agents that inhibit CD47 signaling. In otherexamples, the medium does not include an agent that inhibits CD47signaling. Formation of neural precursor cells or neural cells (forexample after about 1, 2, 3, 4, 5, 6, 7 days or more) can be determinedby morphology (such as neurite formation) or by expression of neuronmarkers (such as MAP2, glial fibrillary acidic protein (GFAP).βIII-tubulin) or astrocyte markers (such as S100b). These cells can bemaintained in culture and passaged multiple times, or can be stored at−80° C. for later use.

In other examples, the iPS or multipotent stem cells or EBs are culturedin a differentiation medium that results in generation of cells havingcharacteristics of mesoderm-derived lineages (such as smooth muscle,endothelial, cartilage, chondrocyte, dermis of skin, connective tissue,urogenital system tissue, heart tissue, hematopoietic, and/or myeloidcells). In one example, iPS cells are transferred into a smooth musclecell differentiation medium, such as Smooth Muscle Basal Medium (forexample, commercially available from Lonza. Basel, Switzerland)supplemented with PDGF (10 ng/ml) and TGFβ1 (5 ng/ml). In some examples,the medium includes one or more agents that inhibit CD47 signaling. Inother examples, the medium does not include an agent that inhibits CD47signaling. In some examples, cells form EBs in 1-2 days, which are thenplated onto gelatin-coated tissue culture dishes in the same medium.Formation of smooth muscle cells can be determined by morphology (suchas presence of typical vascular smooth muscle morphology) or byexpression of smooth muscle cell markers (such as smooth muscle actin).These cells can be maintained in culture and passaged multiple times, orcan be stored at −80° C. for later use.

In another example, iPS cells or multipotent stem cells or EBs aretransferred into a differentiation medium including hematopoietic growthfactors (e.g., as described in Maxhimer et al., Sci. Transl. Med.1:3ra7, 200)). In some examples, the cells are cultured on a semi-solidmedium. Formation of hematopoietic cells can be determined by cellmorphology (such as formation of colonies with phenotypiccharacteristics of myeloid or erythroid cells) or by expression ofhematopoietic cell markers (for example, CD34, CD11a, CD11b, CD117,AML1, CD2, CD3, CD4, CD8, Gr1, Mac1, and/or B220). In a further example,myeloid cells generated as described above can be cultured with amacrophage differentiation medium (such as medium supplemented withmacrophage colony stimulating factor). Macrophages can be identified bycell morphology and expression of macrophage markers (such as Mac-2). Insome examples, the medium includes one or more agents that inhibit CD47signaling. In other examples, the medium does not include an agent thatinhibits CD47 signaling. These cells can be maintained in culture andpassaged multiple times, or can be stored at −80° C. for later use.

In further examples, the iPS or multipotent stem cells or EBs arecultured in a differentiation medium that results in generation of cellshaving characteristics of endoderm-derived lineages (such ashepatocytes, adipocytes, pancreatic beta-cells, gastrointestinal andrespiratory epithelial cells, endocrine secretory cells, bladder and/orurethral epithelial cells). In one example, iPS cells are transferredinto a hepatocyte differentiation medium, such as DMEM with L-glutamine,penicillin/streptomycin and 1% ITS (e.g., commercially available frommultiple suppliers, including Life Technologies. Carlsbad. CA)supplemented with HGF (e.g. 20 ng/ml), Oncostatin M (e.g., 10 ng/ml),and dexamethasone (e.g., 10 nM). In some examples, the medium includesone or more agents that inhibit CD47 signaling. In other examples, themedium does not include an agent that inhibits CD47 signaling. In someexamples, cells form EBs in 1-2 days, which then form hepatocytes.Formation of hepatocytes can be determined by morphology or byexpression of hepatocyte markers (such as α-fetoprotein). These cellscan be maintained in culture and passaged multiple times, or can bestored at −80° C. for later use.

In another example, iPS or multipotent stem cells or EBs are transferredinto a mesenchymal cell differentiation medium, such as such as a basalmedium supplemented with adipogenic factors (e.g., commerciallyavailable from BD Biosciences, ScienCell, or Life Technologies). In someexamples, the medium includes one or more agents that inhibit CD47signaling. In other examples, the medium does not include an agent thatinhibits CD47 signaling. In some examples, cells form EBs in 1-2 days,which then form adipocytes. Formation of adipocytes can be determined bypresence of lipid vacuoles (for example, positive for Oil Red O) or byexpression of adipocyte markers (such as RABP4, adiponectin,adipocytokines, and/or leptin). These cells can be maintained in cultureand passaged multiple times, or can be stored at−80° C. for later use.

One of ordinary skill in the art can identify additional differentiationmedia and cell culture conditions appropriate to differentiate thedisclosed iPS or multipotent stem cells or EBs to other cell types. Thedifferentiation conditions provided herein are exemplary, and should notbe considered to be limiting.

C. Expanding Stem Cells or Differentiated Cells

The disclosed methods include maintaining and/or expanding stem cells ina de-differentiated state capable of self-renewing proliferation bycontinued exposure of the cells to an agent that blocks CD47 signaling.The de-differentiated state is maintained as long as the cells arecultured in appropriate media and exposed to a CD47 blocking agent. Insome embodiments transient exposure to a CD47 blocking agent issufficient to induce this de-differentiated state resulting in cellscapable of self-renewing proliferation. The cells (such as stem cells,for example, induced pluripotent or multipotent stem cells) arecontacted with an agent that blocks CD47 signaling as described aboveand are maintained and passaged in culture utilizing standardtechniques.

In additional embodiments, the disclosed methods include maintainingand/or expanding differentiated cells (such as primary lineage-committedcells or cells differentiated from induced pluripotent stem cells) bycontinued exposure of the cells to an agent that blocks CD47 signaling.The cells are cultured in appropriate media and exposed to a CD47signaling blocking agent, as described above. In some examples, thedifferentiated cells are maintained or expanded in medium includes oneor more agents that inhibit CD47 signaling. In other examples, thedifferentiated cells are maintained or expanded in medium does notinclude an agent that inhibits CD47 signaling.

V. Compositions and Methods for CD47/TSP1 Blockade

The disclosed methods include inhibiting or blocking CD47 signaling(such as CD47/TSP1 signaling), for example to induce formation ofpluripotent stem cells or to generate lineage-committed stem cells. Invarious embodiments, inhibiting CD47 signaling includes one or more ofinhibiting the expression of CD47, inhibiting the expression of TSP1,removing endogenous TSP1 or CD47, or blockading or inhibiting theinteraction between endogenous TSP1 and CD47.

Agents that block or inhibit CD47 signaling include but are not limitedto peptides, antibodies, antisense oligonucleotides, morpholinos, orsmall molecule inhibitors. The agent that inhibits CD47 signalingincludes, in various embodiments, a synthetic peptide having specificbinding affinity for CD47; a synthetic peptide having specific bindingaffinity for TSP1; an oligonucleotide comprising at least about 15contiguous bases and that hybridizes to the mature or unprocessednuclear mRNA of CD47 under high stringency conditions; anoligonucleotide comprising at least about 15 contiguous bases and thathybridizes to the mRNA of TSP1 under high stringency conditions; anisolated or recombinant TSP1 or CD47 molecule or soluble fragmentthereof, or molecule that binds thereto; an agent that decreases theexpression of CD47; an agent that decreases the expression of TSP1; anagent that enhances the proteolysis of CD47; an agent that enhances theproteolysis of TSP1; an agent that enhances removal of CD47 from thecell surface: a CD47 antagonist; an antibody that specifically bindsTSP1; an antibody that specifically binds CD47; or a mixture of two ormore thereof. Exemplary inhibitors of CD47 signaling include thosedescribed in U.S. Pat. No. 8,236,313 and International Pat. Publ. No. WO2010/017332, both of which are incorporated herein by reference in theirentirety.

A. Suppression of Protein Expression

In some embodiments, inhibition or blockade of CD47 signaling isachieved by reducing or suppressing TSP1 or CD47 protein expression, forexample in methods of inducing pluripotent or multipotent stem cells ormethods of generating lineage-committed stem cells or differentiatedcells, such as exemplified herein.

Although the mechanism by which antisense RNA molecules interfere withgene expression has not been fully elucidated, it is believed thatantisense RNA molecules (or fragments thereof) bind to the endogenousmRNA molecules and thereby inhibit translation of the endogenous mRNA,splicing of the nuclear mRNA precursor, or result in its degradation. Areduction of protein expression in a cell may be obtained by introducinginto cells an antisense construct based on TSP1 or CD47 encodingsequences, including the human (or other mammalian) TSP1 cDNA or CD47cDNA or gene sequence or flanking regions thereof. For antisensesuppression, a nucleotide sequence from a TSP1- or CD47-encodingsequence, for example all or a portion of a TSP1 cDNA or gene or all ora portion of a CD47 cDNA or gene, is arranged in reverse orientationrelative to the promoter sequence in the transformation vector. One ofordinary skill in the art will understand how other aspects of thevector may be chosen.

The introduced sequence need not be the full length of the cDNA or gene,or reverse complement thereof, and need not be exactly homologous to theequivalent sequence found in the cell type to be transformed. Generally,however, where the introduced sequence is of shorter length, a higherdegree of homology to the native target sequence will be needed foreffective antisense suppression. The introduced antisense sequence inthe vector may be at least 15 nucleotides in length, and improvedantisense suppression will typically be observed as the length of theantisense sequence increases. The length of the antisense sequence inthe vector advantageously may be greater than about 20 nucleotides,greater than about 30 nucleotides, or greater than about 100nucleotides. For suppression of the TSP1 gene itself, transcription ofan antisense construct results in the production of RNA molecules thatare the reverse complement of mRNA molecules transcribed from theendogenous TSP1 gene in the cell. For suppression of the CD47 geneitself, transcription of an antisense construct results in theproduction of RNA molecules that are the reverse complement of mRNAmolecules transcribed from the endogenous CD47 gene in the cell.

Suppression of endogenous TSP1 or CD47 expression can also be achievedusing ribozymes. Ribozymes are synthetic molecules that possess highlyspecific endoribonuclease activity. The production and use of ribozymesare disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508. The inclusionof ribozyme sequences within antisense RNAs may be used to confer RNAcleaving activity on the antisense RNA, such that endogenous mRNAmolecules that bind to the antisense RNA are cleaved, which in turnleads to an enhanced antisense inhibition of endogenous gene expression.

Suppression can also be achieved using RNA interference, using known andpreviously disclosed methods. Several models have been put forward toexplain RNAi, in particular the mechanisms by which the cleavage derivedsmall dsRNAs or siRNAs interact with the target mRNA and thus facilitateits degradation (Hamilton et al., Science 286:950, 1999: Zamore et al.,Cell 101:25, 2000; Hammond et al., Nature 404:293, 2000; Yang et al.,Curr. Biol. 10:1191, 2000; Elbashir et al., Genes Dev. 15:188, 2001:Bass Cell 101:235, 2000). It has been proposed that the cleavage derivedsmall dsRNAs or siRNAs act as a guide for the enzymatic complex requiredfor the sequence specific cleavage of the target mRNA. Evidence for thisincludes cleavage of the target mRNA at regular intervals of about 21-23nucleotides in the region corresponding to the input dsRNA (Zamore etal., Cell 101, 25, 2000), with the exact cleavage sites corresponding tothe middle of sequences covered by individual 21 or 22 nucleotide smalldsRNAs or siRNAs (Elbashir et al., Genes Dev. 15:188, 2001). Althoughmammals and lower organisms appear to share dsRNA-triggered responsesthat involve a related intermediate (small dsRNAs), it is likely thatthere will be differences as well as similarities in the underlyingmechanism, dsRNAs can be formed from RNA oligomers producedsynthetically (for technical details see material from the companiesXeragon and Dharmacon, both available on the internet). Small dsRNAs andsiRNAs can also be manufactured using standard methods of in vitro RNAproduction. In addition, the Silencer™ siRNA Construction kit (andcomponents thereof) available from Ambion (Catalog #1620; Austin, TX),which employs a T7 promoter and other well-known genetic engineeringtechniques to produce dsRNAs. Double stranded RNA triggers could also beexpressed from DNA based vector systems.

Inhibition also can be accomplished using morpholino oligonucleotides,for instance as described herein. The morpholino can be delivereddirectly to cells (for example, in vitro) or can be administered to asubject as herein described. In particular embodiments, the morpholinois an antisense morpholino oligonucleotide complementary to CD47 (suchas human and/or murine CD47) or TSP1 (such as human and/or murine TSP1).In one non-limiting example is a CD47 morpholino with the nucleic acidsequence CGTCACAGGCAGGACCCACTGCCCA (SEQ ID NO: 35).

The nucleic acids and nucleic acid analogs that are used to suppressendogenous TSP1 or CD47 expression may be modified chemically orbiochemically or may contain one or more non-natural or derivatizednucleotide bases, as will be readily appreciated by those of ordinaryskill in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications, such asuncharged linkages (for example, methyl phosphonates, phosphotriesters,phosphoramidates, carbamates, etc.), charged linkages (for example,phosphorothioates, phosphorodithioates, etc.), pendent moieties (forexample, polypeptides), intercalators (for example, acridine, psoralen,etc.), chelators, alkylators, and/or modified linkages (for example,alpha anomeric nucleic acids, etc.). The term nucleic acid molecule alsoincludes any topological conformation, including single-stranded,double-stranded, partially duplexed, triplexed, hair-pinned, circularand padlocked conformations. Also included are synthetic molecules thatmimic polynucleotides in their ability to bind to a designated sequencevia hydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

Additionally, although particular exemplary sequences are disclosedherein, one of ordinary skill in the art will appreciate that thepresent methods also encompass sequence alterations of the disclosedagents that yield the same results as described herein. Such sequencealterations can include, but are not limited to, deletions, basemodifications, mutations, labeling, and insertions.

Suppression of protein expression may also be achieved through agentsthat enhance proteolysis of CD47 or TSP1 (Allen et al., Endocrinologv150:1321-1329, 2009). In other particular examples, the suppression ofCD47 expression involves an agent that enhances the removal of CD47 fromthe cell surface or decreases the transcription, mRNA processing, ortranslation of CD47. Similar embodiments are envisioned, regardingsuppression of TSP1.

B. Suppression of Protein Activity

In some embodiments, inhibition or blockade of CD47 signaling isachieved by reducing or suppressing TSP1 or CD47 protein activity, forexample in methods of inducing pluripotent or multipotent stem cells ormethods of generating lineage-committed stem cells or differentiatedcells, such as exemplified herein.

In some examples, an inhibitor of CD47 signaling includes an agent thatdecreases or blocks binding of a ligand (such as TSP1) to CD47. Thedetermination that an agent (such as an antibody or a peptide) inhibitsthe association between TSP1 and CD47 may be made, for example, usingassays known to one of ordinary skill in the art. For instance, thedetermination that an agent inhibits TSP1 binding to purified orrecombinant CD47 can be made by comparing the binding activity alonewith the binding activity in the presence of the agent using a solidphase ligand binding assay. An agent that inhibits the activity of TSP1to signal through CD47 on cells will reduce the activity of acGMP-dependent reporter in a suitable transfected cell assay by acertain amount, for example, by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or even by 100%. In addition, an agent that inhibits the activity orCD47 or TSP1 can be identified using any one of the assays describedherein, including, but not limited to, determining c-Myc expression in acell. An agent that inhibits CD47 signaling will increase c-Mycexpression (such as an increase in c-Myc mRNA or c-Myc protein) in acell or population of cells by a certain amount, for example by 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or more ascompared to a suitable control.

Thus, in various embodiments an inhibitor of CD47 signaling includesantibodies (such as monoclonal antibodies or humanized antibodies) thatspecifically bind to CD47 or TSP1. In some examples, an antibody thatspecifically binds CD47 is of use in the methods disclosed herein. Inother examples, an antibody that specifically binds TSP1 is of use inthe methods disclosed herein. Antibodies that specifically bind to CD47or TSP1 include poly clonal antibodies, monoclonal antibodies, orhumanized monoclonal antibodies, or fragments thereof. Methods ofconstructing such antibodies are known in the art (see, for example,Green et al., “Production of Polyclonal Antisera,” in: ImmunochemicalProtocols, pages 1-5, Manson, ed., Humana Press, 1992: Coligan et al.,“Production of Polyclonal Antisera in Rabbits. Rats, Mice and Hamsters,”in: Current Protocols in Immunology, section 2.4.1, 1992; Kohler &Milstein, Nature 256:495, 1975; Coligan et al., sections 2.5.1-2.6.7;and Harlow et al. in: Antibodies: a Laboratory Manual, page 726, ColdSpring Harbor Pub., 1988). In addition, such antibodies may becommercially available. In some examples, an inhibitor of CD47 signalingincludes an anti-CD47 antibody, such as anti-CD47 antibodies B6H12, BRIC126, 6H9, Clkm1, OVTL16, OX101, mIAP410, or mIAP301 (also referred to asab301), a binding fragment of any one of these, or a humanized versionof any one of these, or an antibody or fragment thereof that competeswith B6H12, BRIC 126, 6H9, Clkm1, OVTL16, OX101, mIAP410, or mIAP301 forbinding. In other examples, an inhibitor of CD47 signaling includes ananti-TSP1 antibody, such as C6.7, HB8432, D4.6, A65M, A4.1, A6.1, orSPM321, a binding fragment of any one of these, or a humanized versionof any one of these, or an antibody or fragment thereof that competeswith C6.7, HB8432, D4.6, A65M, A4.1, A6.1, or SPM321 for binding. It isto be understood that CD47 signaling inhibitors for use in the presentdisclosure also include novel CD47 or TSP1 antibodies developed in thefuture.

In other embodiments, an inhibitor of CD47 signaling includes a peptidethat specifically binds to CD47 or TSP1. In some examples an inhibitorof CD47 signaling is a CD47-binding peptide, such as a TSP1-derivedCD47-binding peptide. Exemplary CD47-binding peptides include 7N3(FIRVVMYEGKK; SEQ ID NO: 1) and 4N1 (also known as 459; RFYVVMWK; SEQ IDNO: 37). Additional CD47-binding peptides include those described inU.S. Pat. No. 8,236,313, incorporated herein by reference in itsentirety. It is to be understood that CD47 signaling inhibitors for usein the present disclosure also include novel CD47 or TSP1 bindingpeptides developed in the future.

In additional embodiments, an inhibitor of CD47 signaling includes asmall molecule (such as a small organic molecule). Some small moleculeinhibitors may inhibit CD47 or TSP1 expression or activity. It is to beunderstood that CD47 signaling inhibitors for use in the presentdisclosure also include novel CD47 or TSP1 small molecule inhibitorsdeveloped in the future.

VI. Therapeutic Uses

The methods disclosed herein can be used for the ex vivo generationand/or expansion of induced pluripotent or multipotent stem cells orlineage-committed (differentiated cells) for cell-based therapies andtissue engineering. The disclosed methods have several advantages overcurrent methods of generating immortalized cells. The disclosed methodsdo not increase risk of malignant transformation of the cells, forexample, because they do not use transformation of the cells (such aswith T antigen) or telomerase. In addition, in at least someembodiments, the disclosed methods do not require use of bacterial orviral vectors for creating continuously proliferating cells. Thedisclosed methods also are more suitable for clinical uses because theyutilize defined molecular entities. In other embodiments, the methodsinclude administering an inhibitor of CD47 signaling to a subject.

Administration to cells of inhibitors of CD47 signaling can be local orsystemic. Examples of local administration include, but are not limitedto, topical administration, subcutaneous administration, transdermaladministration, intramuscular administration, intrathecaladministration, intrapericardial administration, intra-ocularadministration, topical ophthalmic administration, or administration tothe nasal mucosa or lungs by inhalational administration. In addition,local administration includes routes of administration typically usedfor systemic administration, for example by directing intravascularadministration to the arterial supply for a particular organ. Thus, inparticular embodiments, local administration includes intra-arterialadministration and intravenous administration when such administrationis targeted to the vasculature supplying a particular organ. Localadministration also includes the incorporation of active compounds andagents into implantable devices or constructs, such as vascular stentsor other reservoirs, which release the active agents and compounds overextended time intervals for sustained treatment effects.

Systemic administration includes any route of administration intended todistribute an active compound or composition widely throughout the body,for example, via the circulatory system. Thus, systemic administrationincludes, but is not limited to intra-arterial and intravenousadministration. Systemic administration also includes, but is notlimited to, topical administration, subcutaneous administration,transdermal administration, intramuscular administration, oradministration by inhalation, when such administration is directed atabsorption and distribution throughout the body by the circulatorysystem. Systemic administration also includes oral administration, insome examples.

In some embodiments, induced pluripotent or multipotent stem cells aregenerated by contacting primary cells with an agent that blocks CD47signaling as described above, and increased numbers of these stem cellscan be obtained by continuous culture of the cells with an agent thatblocks CD47 signaling to obtain the desired number of cells. Similarly,a population of differentiated cells of a desired cell type can begenerated by contacting primary cells with an agent that inhibits CD47signaling, followed by culture in an appropriate differentiation medium,as described above, and increased numbers of these differentiated cellscan be obtained by continuous culture of the cells with an agent thatblocks CD47 signaling to obtain the desired number of cells.

In some embodiments, the resulting cells can be utilized for ex vivotissue engineering applications. For example, the disclosed methodsinclude increasing cell population of a tissue matrix by contacting thetissue matrix with cells and an inhibitor of CD47 signaling ex vivo. Insome examples, the cells could be used to populate or repopulate atissue matrix, for example a decellularized organ or natural tissuematrix or to populate a synthetic organ or synthetic tissue scaffold.Methods of preparing a decellularized tissue matrix are known to one ofordinary skill in the art (see. e.g., Gilbert, J. Cell. Biochem.113:2217-2222, 2012, incorporated herein by reference). For example theiPS cells could be perfused into a decellularized organ or tissue matrixor a synthetic organ or tissue scaffold under conditions sufficient topermit seeding of the matrix or scaffold with the iPS cells and then toproliferate and differentiate to form a bioengineered organ. In someexamples, cells (such as iPS cells) would be perfused into the matrix orscaffold in the presence of the CD47 signaling inhibitor, for example toseed the matrix, and then the matrix or scaffold would be perfused withan appropriate differentiation medium (which optionally may also includea CD47 signaling inhibitor), for example to permit differentiation ofthe cells. These methods could be used to bioengineer entire organs(such as a liver, kidney, heart, lung, bladder, trachea, or esophagus),which could then be transplanted into a subject in need of an organtransplant. These methods can also be used to bioengineer tissues orportions of organs, such as vessels for vascular graft, lymphatics,replacement heart valves, skin grafts, bone grafts, joint components(such as the femoral head), airways, urethra, pancreatic islets, nerves,cornea, retina, inner ear, cardiac muscle, or to replace cartilaginoustissue (such as in the trachea), which could then be transplanted into asubject in need of the organ or tissue. In some instances anydecellularized matrix, natural or synthetic, can be combined with a CD47signaling inhibitor agent, removing the barrier to cell invasion of,migration through, and restoration of the complex 3D structure. Inparticular examples, the methods increase cell population of a tissuematrix by at least about 10% (such as at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or more) as compared to a tissue matrixcontacted with cells but not contacted with an inhibitor of CD47signaling.

In additional embodiments, the methods disclosed herein can be used togenerate and/or expand populations of cells for administration to asubject. In one example, the disclosed methods could be used to expandpancreatic cells (such as islet cells), which could then be transplantedinto a subject in need of pancreatic cells (for example, a subject withdiabetes). In other examples, the disclosed methods could be used togenerate and expand populations of hematopoietic cells (such ashematopoietic stem cells or myeloid- or lymphoid-committed cells) for asubject in need of such cells. In some examples, hematopoietic stemcells can be generated and/or expanded for bone marrow transplantationto a subject with cancer or a subject with an immune-deficiency or fortreating a subject with radiation toxicity. Without being limited bytheory, it is believed that by enhancing the potential of a marrowtransplant by the disclosed methods may allow for decreased amount ofmarrow harvested and may increase the success rate of transplant. In yetother examples, the disclosed methods could be used to expand cytotoxicT cells for adoptive immunotherapy in subjects with cancer. One ofordinary skill in the art can select appropriate cell types and cellnumbers to be administered to a subject to treat or inhibit a condition.In some examples, the methods include administering the cells to asubject by local administration (such as transplantation or injectioninto a tissue or organ) or systemic administration (such as intravenousadministration). In other examples, the cells are administered to asubject by subcutaneous or transdermal administration, for example byinjection into or under the skin. See, e.g., U.S. patent publications2011/0110898, 2011/0274665, 2009/0130066, 2008/0311089, 2007/0207131,2007/0154462, 2007/0154461, 2006/0039896, 2005/0271633, 2005/0186149,2003/0228286, and 2002/0197241; as well as U.S. Pat. Nos. 5,591,444,5,660,850, 5,665,372, and 5,858,390.

In other embodiments, the methods include administering an inhibitor ofCD47 signaling to a subject, for example, to increase or generateinduced pluripotent stem cells in vivo. The resulting iPS cells couldfor example repopulate damaged tissue (such as a wound or burn or afractured bone) or enhance the effectiveness of a bone marrow celltransplant in a subject. Compositions including inhibitors of CD47 andtheir administration are described in U.S. Pat. No. 8,236,313 and U.S.Pat. Publ. Nos. 2011/013564; incorporated herein by reference.

In some embodiments, a CD47 signaling inhibitor (such as a peptide,antibody or antibody fragment, nucleic acid, or inhibitoryoligonucleotide (e.g., morpholino)) is administered locally to anaffected area, for example by direct administration to a wound or othersite in which recruitment or generation of iPS is desired (e.g. pancreasor bone marrow), or is incorporated into an implant device and placeddirectly at an affected area, such as a wound or other tissue injury. Insome embodiments, administration is, for example, by direct topicaladministration to a wound, or by intra-arterial, intravenous,subcutaneous, or intramuscular injection into the affected area.Efficacy of the treatment is shown, for example, by a regression ofsymptoms, for example wound healing or generation of new tissue or byincreased skin temperature or a color change in the skin of the limbs.For subjects with a wound such as a burn or a graft, administration is,for example, by subcutaneous or intravenous injection, by directinjection of the wound or burn or graft bed, or by topical application.Efficacy of the treatment is determined, for example, by an improvementin wound healing.

In additional examples, administration of an inhibitor of CD47 signalingcan be administered to enhance healing in conditions of delayed healing,such as non-union bone fractures, chronic wounds, or non-healing tendoninjuries, for example by direct topical administration near or to awound, or by intra-arterial, intravenous, subcutaneous, or intramuscularinjection into the affected area.

In further examples, an effective amount of an inhibitor of CD47signaling may be utilized to treat or prevent hair loss. The inhibitorof CD47 signaling is administered topically to an affected area (such asthe scalp) or is administered transdermally or subcutaneously to anaffected area, or optionally systemically.

In additional examples, iPS or multipotent stem cells or differentiatedcells prepared according to the methods described herein or CD47inhibitors may be administered to the eye (for example administered orimplanted intravitreally) to treat a subject with vision loss. In someexamples, the subject has a progressive vision disorder, such as retinaldegeneration (for example, retinitis pigmentosa), macular degeneration,or glaucoma. Cells or CD47 inhibitors can be administered to the eyetopically, for example topical preparations can include eye drops,ointments, sprays, patches and the like. Cells or compositions can alsobe included in an inert matrix for either topical application orinjection into the eye, such as for intravitreal administration.Liposomes, including cationic and anionic liposomes, can be made usingstandard procedures as known to one skilled in the art. Liposomes can beapplied topically, either in the form of drops or as an aqueous basedcream, or can be injected intraocularly. The cells or CD47 inhibitorscan also be included in a delivery system that can be implanted atvarious sites in the eye, depending on the size, shape and formulationof the implant, and the type of transplant procedure. The deliverysystem is then introduced into the eye. Suitable sites include but arenot limited to the anterior chamber, anterior segment, posteriorchamber, posterior segment, vitreous cavity, suprachoroidal space,subconjunctiva, episcleral, intracorneal, epicorneal and sclera.

An effective amount of a therapeutic CD47 inhibitor (such as a peptide,antibody, inhibitor peptide-encoding DNA, or oligonucleotide (e.g.,morpholino)) can be administered in a single dose, or in multiple doses,for example daily, weekly, every two weeks, or monthly during a courseof treatment. Additionally, the therapeutic agents may be incorporatedinto or on implantable constructs or devices, such as vascular stents,for sustained regional or local release.

In some examples, the methods include identifying or selecting a subjectfor administration of a CD47 signaling inhibitor or iPS or multipotentstem cells or differentiated cells prepared according to the methodsdescribed herein. For example, the methods include selecting a subjectwith damaged tissue (such as a burn, broken bone, wound, or other tissuedamage), a subject in need of a bone marrow cell transplant (such as asubject with a hematological cancer or immune-deficiency or radiationtoxicity), or a subject with diabetes and administering an inhibitor ofCD47 signaling or iPS or multipotent stem cells or differentiated cellsprepared according to the methods described herein to the selectedsubject. In other examples, the methods include selecting a subject withhair loss (such as alopecia, or radiation-induced alopecia) andadministering an inhibitor of CD47 signaling or iPS or multipotent stemcells or differentiated cells prepared according to the methodsdescribed herein to the selected subject. In still further examples, themethods include selecting a subject with vision loss (such as retinaldegeneration, macular degeneration, or glaucoma) and administering aninhibitor of CD47 signaling or iPS or multipotent stem cells ordifferentiated cells prepared according to the methods described hereinto the selected subject.

VII. Kits

Also disclosed herein are kits that can be used to inducelineage-committed, pluripotent, or multipotent stem cells from primarycells, generate differentiated cells from primary cells, and/or expandstem cells or lineage-committed differentiated cells in culture. In someembodiments, the kit includes one or more agent that blocks CD47signaling, such as one or more of an anti-CD47 antibody or fragmentthereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, aCD47 morpholino, an anti-TSP1 antibody or fragment thereof, aTSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1morpholino. In other embodiments, the kit includes a small moleculecapable of binding to CD47 or a small molecule capable of binding toTSP1.

In one example, the kit includes a CD47 morpholino, such as a morpholinoincluding the sequence of SEQ ID NO: 35. In another example, the kitincludes an anti-CD47 antibody or fragment thereof, such as monoclonalantibody MIAP301, monoclonal antibody OX101, or monoclonal antibodyB6H12. In a further example, the kit includes a CD47 binding peptide,such as a peptide including the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 37. In another example, the kit includes an anti-TSP1antibody or fragment thereof, such as monoclonal antibody A6.1 ormonoclonal antibody C6.7.

The kits may further include additional components such as instructionalmaterials and additional reagents, for example cell culture medium (suchas growth medium or differentiation medium) for one or more cell types.In some examples, the kits may include one or more primary cell types(for example, HUVEC). The kits may also include additional components tofacilitate the particular application for which the kit is designed (forexample tissue culture plates). The instructional materials may bewritten, in an electronic form (such as a computer diskette or compactdisk), or may be visual (such as video files).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1: CD47 Inhibits Self-Renewal and Reprogramming byRegulating c-Myc and Other Stem Cell Transcription Factors

This example shows that primary cells obtained from CD47- orthrombospondin-1-null mice lack the rapid senescence in culturetypically observed for wild type (WT) primary mouse cells, and that theresilience of these null cell primary cultures derives at least in partfrom enhanced self-renewal and an ability to undergo stem cellreprogramming.

INTRODUCTION

CD47 is a signaling receptor for the secreted matricellular proteinthrombospondin-1 and the counter-receptor for signal-regulatoryprotein-α (SIRPα), which on phagocytic cells recognizes CD47 engagementas a marker of self (Matozaki et al., Trends Cell Biol 19, 72-80, 2009;Roberts et al., Matrix Biol., 31(3):162-169, 2012: Frazier et al., inNature Signaling Gateway, doi:10.1038/mp.a002870.01, 2010). Mice lackingCD47 or thrombospondin-1 are profoundly resistant to several types oftissue stress including ischemia, ischemia/reperfusion, and high doseirradiation (Roberts et al., Matrix Biol., 31(3):162-169, 2012; Isenberget al., Blood 109, 1945-1952, 2007; Thakar et al., J Clin Invest 115,3451-3459, 2005; Isenberg et al., Surgery 144, 752-761, 2008; Isenberget al., Am. J. Pathol. 173, 1100-1112, 2008). The survival advantage ofischemic CD47-null tissues is mediated in part by increased nitricoxide/cGMP signaling 2, but this pathway is not sufficient to accountfor the resistance to ionizing radiation caused by CD47 blockade(Maxhimer et al., Sci. Transl. Med. 1:3ra7, 2009). Radioresistanceassociated with CD47 blockade is cell autonomous (Id.), indicating thatadditional pro-survival signaling pathways are controlled by CD47.

Engaging CD47 in some cell types triggers apoptosis or type IIIprogrammed cell death (Frazier et al., in Nature Signaling Gateway,doi:10.1038/mp.a002870.01, 2010; Bras et al., Mol Cell Biol 27,7073-7088, 2007). BCL2/adenovirus E1B 19 kDa protein-interacting protein3 (BNIP3) is a pro-apoptotic BH3 domain protein that was identified asan interacting partner with the cytoplasmic tail of CD47 and implicatedin CD47-dependent cell death (Lamy et al., J Immunol 178, 5930-5939,2007). Furthermore, localization of the dynamin-related protein Drp1 isregulated by CD47 ligation and was implicated in the control ofmitochondria-dependent death pathways by CD47 (Bras et al., Mol CellBiol 27, 7073-7088, 2007). Drp1 mediates mitochondrial fission (Kageyamaet al., Curr Opin Cell Biol 23, 427-434, 2011). Correspondingly, sometissues in CD47-null and thrombospondin-1-null mice show increasedmitochondrial numbers and function (Frazier et al., Matrix Biol 30,154-161, 2011). Although these studies provide some insights into howCD47 ligation can trigger cell death, regulation of mitochondrialfunction is unlikely to account for the profound resistance to stressconferred by the absence or blockade of CD47 signaling.

In contrast to the above noted survival advantages of cells lacking orexpressing decreased levels of CD47, elevated expression of CD47 canconfer an indirect survival advantage in vivo. CD47 engages SIRPα onmacrophages and prevents phagocytic clearance by conveying a “don't eatme” signal (Matozaki et al., Trends Cell Biol 19, 72-80, 2009). Tyrosineresidues in the cytoplasmic domain of SIRPα become phosphorylated inresponse to engaging CD47 and modulate the recruitment and/or activityof several signaling molecules including SHP1, SHP2, SKAP55hom/R,FYB/SLAP-130, and PYK2 (Id). Thus, erythrocytes lacking CD47 expressionare rapidly cleared in vivo (Oldenborg et al., Science,288(5473):2051-2054, 2000). Similarly, elevated expression of CD47 onseveral types of cancer cells has been shown to inhibit their killing bymacrophages or NK cells (Chan et al., Proc Natl Acad Sci USA,106(33):14015-10421, 2009; Kim et al., 2008; Majeti et al., Cell138(2):286-299, 2009). Conversely, CD47 antibodies that block SIRPαbinding enhance macrophage-dependent clearance of tumors in severalmouse models (Chao et al., Cancer Res. 71(4):1374-1384, 2011; Chao etal., Cell 142(5):699-713, 2010; Majeti et al., Cell 138(2):286-299,2009; Willingham el al., Proc Natl Acad Sci US4, 109(17):6662-6667,2012), although others have shown that such clearance can occurindependent of inhibitory SIRPα signaling (Zhao et al., EMBO Rep.12(6):534-541, 2011).

Taken together, these studies indicate two opposing roles for CD47 incell survival. The cell autonomous advantages of decreased CD47expression, leading to less inhibitory CD47 signaling, must be balancedagainst the need to maintain sufficient CD47 levels to preventphagocytic clearance in vivo. Hematopoietic stem cells also exhibitelevated CD47 expression, and high CD47 expression in the stem cellniche was proposed to be important to protect stem cells from innateimmune surveillance (Jaiswal et al., Cell 138(2):271-285, 2009).

Methods

Cell culture and reagents: Thrombospondin-1 null (Lawler et al., J ClinInvest 101, 982-992, 1998) CD47 null mice (Lindberg et al., Science 274,795-798, 1996) extensively back-crossed onto a C57Bl/6J background andWT mice were maintained in a pathogen-free environment according toprotocols approved by the NCI Animal Care and Use Committee. Mouse lungendothelial cells were isolated and their purity verified as describedpreviously (Zhou et al., Oncogene 25, 536-545, 2006). These conditionswere previously documented to reproducibly yield >95% pure endothelialcells at passage two (CD31+, smooth muscle actin-). Mouse lungendothelial cells were cultured at 37° C. with 5% CO₂ using EndothelialGrowth Medium-2 (EGM2) (Thermo Scientific Fisher, Inc., Waltham, MA).Cell populations from mouse spleens were separated using the Pan T cellIsolation (130-095-130). CD4(L3T4) (130-049-201), CD8a(Ly-2)(130-049-401) CD11b (130-049-601), CD19 microbead kits (130-052-201)(MACS, Miltenyi Biotech Germany).

V6.5 mouse ES cells were cultured on gelatin-coated dishes with mouseembryonic fibroblast (MEF) feeder cells using standard mouse ES mediumcontaining DMEM (high glucose), 15% ES cell-qualified FBS, 200 mML-glutamine, non-essential amino acids (Life Technologies), Pen/Strep,0.1 mM 2-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor(LIF).

The thrombospondin-1-derived CD47-binding peptide 7N3(1102-FIRVVMYEGKK-1112; SEQ ID NO: 1) and a corresponding inactivecontrol peptide 604 (FIRGGMYEGKK; SEQ ID NO: 2) were synthesized byPeptides International (Louisville, KY) (Barazi et al., J Biol Chem 277,42859-42866, 2002). Human TSP1 was purified from the supernatant ofthrombin-activated platelets obtained from the NIH Blood Bank aspreviously described (Roberts et al., J Tissue Cult Methods 16, 217-222,19). A somatic mutant of the Jurkat human T lymphoma cell line lackingCD47, JinB8, was provided by Dr. Eric Brown (Reinhold et al., IntImmunol 11:707-718, 1999). Jurkat T cells, JinB8. Raji human Burkitt'slymphoma cells with c-Myc under the control of an IgH enhancer, B16 F10murine melanoma, and Rat1 fibroblasts expressing the conditional c-Mycfusion protein (MycER™; Littlewood et al., Nucleic Acids Res 23,1686-1690, 1995) were cultured using RPMI 1640 medium containing 10%FBS, penicillin/streptomycin, and glutamine (Invitrogen, Rockville, MD).

RNA extraction and Real Time PCR: Total RNA was extracted using TRIzol®reagent (Invitrogen. Rockville. MD) 24-36 hours after transfection or asindicated. Harvested mouse tissues were frozen in liquid nitrogen orplaced into RNA Later™ RNA Stabilization Reagent (Ambion. LifeTechnologies. Grand Island, NY). Whole organs (lungs, spleen, kidney,testis, skeletal muscle, brain, heart, and liver) were homogenized inTRIzol® reagent, and RNA was isolated. cDNA was prepared using FirstMaxima First Strand cDNA Synthesis kit for RT-qPCR (Fermentas LifeSciences, Glen Burnie, MD). Real Time PCR was performed using theprimers listed herein as SEQ ID NOs: 3-34, and SYBR Green PCR masterreaction mix (AB applied Sciences, Life Technologies, Grand Island. NY)on an MJ Research OPTICON I instrument (Bio-Rad) with the followingamplification program: 95° C. for 15 minutes, followed by 40 cycles of95° C. for 15 seconds, 58° C. for 20 seconds, 72° C. for 25 seconds, and7° C. for 1 minute. Melting curves were performed for each product from30° C. to 95° C. reading every 0.5° C. with a 6-second dwell time. Thefold changes in mRNA expression were calculated by normalizing tohypoxanthine phosphoribosyltransferase (HPRT1) and TATA-box bindingprotein associated factor (TAF9) for mouse tissues and endothelialcells, or β-2 microglobulin (B2M) mRNA levels for spleen and isolatedsplenocytes. B2M was used for normalization of mRNA levels in humancells. Note that the total RNA yield per cell was higher for allCD47-null and CD47-deficient cells and tissues as compared to WT. Equalamounts of total RNA from WT and CD47 null mouse correspondingly showeddifferences expression for many housekeeping genes, but the above notedreference genes showed minimal differences in Ct values.

Microarray processing: Samples were prepared according to Affymetrixprotocols (Affymetrix, Inc.). RNA quality and quantity were ensuredusing the Bioanalyzer (Agilent Technologies) microfluidics-basedplatform and NanoDrop (Thermo Fisher Scientific, Inc.) micro-volumespectrophotometer, respectively. Per RNA labeling, 300 nanograms oftotal RNA was used in conjunction with the Affymetrix recommendedprotocol for the GeneChip 1.0 ST chips.

The hybridization cocktail containing the fragmented and labeled cDNAswere hybridized to Affymetrix Mouse GeneChip® 1.0 ST chips. The chipswere washed and stained by the Affymetrix Fluidics Station using thestandard format and protocols as described by Affymetrix. The probearrays were stained with streptavidin phycoerythrin solution (MolecularProbes, Carlsbad, CA) and enhanced by using an antibody solutioncontaining 0.5 mg/mL of biotinylated anti-streptavidin (VectorLaboratories, Burlingame, CA). An Affymetrix Gene Chip Scanner 3000 wasused to scan the probe arrays. Gene expression intensities werecalculated using GeneChip® Command Console® Software (AGCC) andExpression Consoler™ Software. CEL files generated by the AffymetrixAGCC program were imported in the Partek Genomic Suite software and RMA(Robust Multichip Analysis) normalization, log 2 transformation andprobe summarization was performed. Anova pairwise comparisons and PCA(Principle Component Analysis) were performed within Partek GenomicSuite. The GEO accession numbers for the microarray data is GSE43133.

GeneSet Enrichment Analysis (GSEA) was used to test whether anestablished gene signature was significantly enriched for genesdifferentially expressed between WT, CD47 null, CD47 null EB-likeclusters, and established embryonic stem cell lines. Description of theGeneSet enrichment analysis (GSEA) and the MSigDB can be found atwww.broadinstitute.org/gsea/.

Teratoma Formation: The v6.5 mouse ES cell line was used as a positivecontrol for testing teratoma formation. These mES cells were cultured inDMEM medium containing 15% fetal bovine serum and 1000 IU/ml LIF(Leukemia Inhibitory Factor). For teratoma formation, the mES cells orCD47−/− endothelial cells were trypsinized, washed once in PBS, andfinally resuspended in PBS at 5×106/ml for mES and 1×107/ml for CD47−/−.The cells suspension was chilled on ice and then mixed with 50% volumeof cold Matrigel (40° C.). The cell-Matrigel mix was draw into a cold 1ml syringe, and 0.15 ml was quickly injected subcutaneously intoNOD.Cg-Prkdcscid II2rgtm1 Wjl/SzJ mice near the region where the hindthigh and the abdomen meet. Therefore, about 5×10⁵ mES cells or 1×10⁶CD47−/− cells from EB-like clusters were injected at each site. Twoweeks after the injection, the mice were observed daily for tumorgrowth. When the tumor reached 2 cm in length, the mouse was euthanized,and the tumors were dissected out for morphological observation.

Colony forming assay: Semisolid medium was prepared based on a previousmethod to quantify embryonic stem cell embryoid body formation (Stenberget al., Cytotechnology 63, 227-237, 2011). Briefly, 1.5% Noble agar wasautoclaved in DMEM low glucose medium. Glutamine and 2% FBS serum wereadded and kept warm at 50° C. A 1.5 ml volume was allowed to solidify ineach Petri dish at RT for 15 min. The mouse lung endothelial cells weretrypsinized and 200.000 cells/ml were suspended in EGM2 medium. For thetop layer, 1.5% agar was diluted to 0.5% with 2xEGM2 medium, and 100 μlper ml cells were added, mixed very quickly, and 1.5 ml was poured onthe top of the base agar layer. Fresh EGM2 medium (1.5 ml) was addedafter 20 days. Colony morphologies were scored after four weeks.

Cell culture medium for macrophage differentiation: Mouse L929 cells (akind gift from Alan Sher, NIH) were grown in DMEM Growth medium (DMEMwith high glucose, 10% FBS, 2 mM L-Glutamine, Penicillin-Streptomycin:all from Life Technologies) at 37° C. under 5% CO₂ until 100% confluent.Conditioned medium was harvested and stored at −80° C.

CD47 deficient mouse cells were either cultured in the presence ofEndothelial Basal Medium −2 (Lonza) or in the presence of 30% L929conditioned medium in RPMI Growth Medium (RPMI 1640, 10% FBS, 2 mML-Glutamine, Penicillin-Streptomycin; all from Life Technologies). Cellswere cultured for ten days at 37° C. under 5% CO₂. The macrophage markerwas tested using Flow Cytometry.

Antibodies/Reagents for Flow Cytometry: Anti-mouse CD11c PE-Cy7, CD11bPE, and B220 PE were all purchased from BD Biosciences (San Jose, CA).Anti-mouse Ly-6C eFluor 450, Ly-6G PerCP-Cy5.5, and CD3e FITC were allpurchased from eBioscience (San Diego, CA). Anti-Mouse Sca-1 PE-Cy5 wasa kind donation from Thomas B. Nutman (NIH). Anti-mouse CD14 APC-Cy7,CD31 AlexaFluor647, CD64 APC, and anti-mouse/human Mac-2 PE werepurchased from BioLegend (San Diego, CA). All flow cytometry antibodieswere titrated for optimal performance. Anti-Rat/Anti-Hamster Ig κcompensation particles were purchased from BD Biosciences.

All cells were dislodged by incubating with Versene solution (LifeTechnologies) and then scraping. They were collected on ice and washedwith buffer (PBS with 3% BSA; Life Technologies). All following stainingsteps were performed on ice and incubated in the dark. After washesbuffer was decanted and cells were stained with all antibodies or eachflorescence minus one control. Compensation beads were used for singlecolor controls, when possible, as directed by the manufacturer.Otherwise, single color controls were made using a mixture of cell.Cells and beads were washed thoroughly prior to acquisition. Data wasacquired using a LSRII (BD Biosciences) and BD FACSDiva™. Software. Datawas analyzed using FlowJo software (Tree Star, Inc., Ashland. OR).

Cell culture medium for neural differentiation: CD47 null mouse lungendothelial cells passaged for six months were seeded into six-welltissue culture plates using basal EBM medium supplemented with FGF2 andEGF (5-20 ng/ml), heparin and gentamycin sulfate. Embryoid bodiesappeared after 24-36 hours. The cells were then plated onto non-tissueculture dishes in heparin-free differentiation medium. Neural precursorcells were visible after six days.

Cell culture medium for smooth muscle cell differentiation: CD47 nullmouse lung endothelial cells were plated into six-well tissue cultureplates using Smooth Muscle Basal Medium (Lonza) supplemented with PDGF(10 ng/ml) and TGF-β1 (5 ng/ml). The embryoid bodies (EBs) wereharvested and transferred to 1% gelatin (Sigma) coated plates. The EBsdifferentiated into smooth muscle cells after six days. Thedifferentiated smooth muscle cells were stained for smooth muscle actin.

Cell culture medium for hepatocyte cell differentiation: Wild type andCD47 null endothelial cells were grown in DMEM+glutamine+p/s+1% ITS(Invitrogen)+HGF (R&D—20 ng/ml), Oncostatin M (R&D ng/ml), 10 nMdexamethasone (Waco Pure Chemical Industries Ltd, Osaka, Japan) withslight modification of Ishkitiev et al., J. Breath Res. 6:017103, 2012.The embryoid bodies were stained for the hepatocyte marker AFP after 36hours.

Cell culture medium for mesenchymal cell differentiation: The WT andCD47 null endothelial cells were grown in BD Mosaic™ hMSC SF culturemedium along with BD Mosaic™ hMSC SF supplement (BD Biosciences). CD47null cells formed embryoid bodies after 36 hours. The embryoid bodieswere collected and differentiated by coating plates with BD Mosaic™ hMSCSF surface (BD Biosciences). For direct transdifferentiation, the plateswere coated with BD Mosaic™ hMSC SF surface according to manufacturer'sinstructions. WT and CD47 null endothelial cells were directly plated oncoated 6-well plates (BD biosciences). The trans-differentiated cellswere stained using oil red after 10-days.

Oil Red O staining for mesenchymal adipocytes: Stock solution of Oil RedO (30) mg of oil red powder+100 ml of isopropanol) was prepared the daybefore staining according to the manufacturer's instructions. For aworking solution, 3 parts of stock solution of Oil Red O and 2 parts ofdeionized water were mixed. The working solution was incubated for 10minutes at RT and filtered with Whatman filter paper several times. Thedifferentiated embryoid cells were cultured in 12-well plates for10-days. To assess adipogenic phenotype, cells were washed with 1×DPBSand fixed with 1-2% Formalin overnight at 4° C. The formalin was removedfrom the wells, and the cells were washed with deionized water. Two mlof 60% of isopropanol was added to each well for 5 minutes. The cellswere then incubated with 2 ml of Oil Red O solution for 5 minutes. Thecells were rinsed with deionized water until clear. A 2 ml volume ofhematoxylin stain was added for 1 minute and then washed with waterimmediately. The wells were covered with water, and images were takenusing phase contrast illumination.

Immunostaining of embryoid bodies and differentiated cells: EBs wereplaced on poly-D lysine coated Lab-Tek cover glass chambers and fixedwith 4% paraformaldehyde for five minutes. EBs were gently washed with1×PBS and permeabilized using 0.3% Triton X-100. The EBs were washed andblocked with 3% BSA for one hour. Primary SOX2 (Abcam) and nestinantibodies (Covance) (1:500) were used for immunostaining.

Differentiated neural cells were cultured overnight using Lab-Tek coverglass 4-well chambers. The cells were washed twice with 1×PBS, fixedusing 4% paraformaldehyde for 5 min, and washed three times. The cellswere permeabilized using 0.3% Triton X-100 in PBS. The cells were washedthree times 5 minutes each and blocked with 5% BSA for one hour. Primaryantibodies against GFAP (DAKO), S100b (Abcam), MAP2, beta tubulin IIIand smooth muscle actin (Sigma) were used. Secondary antibodies (AlexaFluor® 488 Goat Anti-Mouse IgG1 or Alexa Fluor® 488 Goat Anti-RabbitIgG. Invitrogen) were used. Confocal images were captured using Zeiss710 Zeiss AIM software on a Zeiss LSM 710 Confocal system (Carl ZeissInc., Thornwood, NY) with a Zeiss Axiovert 100M inverted microscope and50 mW argon UV laser tuned to 364 nm, a 25 mW Argon visible laser tunedto 488 nm and a 1 mW HeNe laser tuned to 543 nm. A 63× Plan-Neofluar 1.4NA oil immersion objective was used at various digital zoom settings.

Immunostaining and differentiation of cystic embryoid bodies: CD47 nullcell embryoid bodies were collected and transferred to gelatin coatedT185 flask (Nunc) using RPMI complete media for 6 days. The embryoidbodies differentiated into heterogeneous colonies. The individualcolonies were picked and transferred further into gelatin coated Willicodish. The colonies were cultured using appropriate differentiation media(neural smooth muscle, and hepatocyte) for 36 hours. The embryoid bodieswere fixed with 4% PFA for 1-2 h at RT. The embryoid bodies were washedthree times with 1×PBS (without Ca and Mg ions). The embryoid bodieswere blocked with blocking buffer (3% BSA in PBS+0.2% Triton® X-100) for1-2 hours. The primary antibodies (1:100 in blocking buffer) for neural(ectoderm), smooth muscle actin (mesoderm) and Alpha-fetoprotein(endoderm) markers used overnight at 4° C. The embryoid bodies werewashed with blocking buffer three times. Secondary antibodies (1:1000ratios of Alexa Fluor® 488 Goat Anti-Mouse IgG1 or Alexa Fluor® 488 GoatAnti-Rabbit IgG. Invitrogen) were used. The embryoid bodies were washedthree times with 1×PBS. Embryoid bodies were dried using Kimwipes.VECTASHIELD from Vector Laboratories (Burlingame, CA) with DAPI used formounting. The confocal images were captured using Zeiss 710 Zeiss AIMsoftware on a Zeiss LSM 710 Confocal system as above mentioned. TheZ-stack images were captured and exported as an Avi File using the ZENsoftware.

Sox2 immunohistochemistry: Lung and spleen tissues from WT and CD47−/−mice were fixed in 10% formalin. Tissue was paraffin embedded and cutinto 5 μm thick sections. Immunostaining was performed using an antibodyto SOX2 (1:100) or a non-specific control antibody and detected usingthe DAKO LASB Universal Kit. Stained sections were visualized andphotographed under light microscope using the Q-Imaging system.

Western Blots: Equal number of lung endothelial cells from WT and CD47null were plated in six-well plates overnight. Cell lysates were madefrom washed cells using NP-40 lysis buffer (50 mM Tris pH 8.0, 150 mMNaCl and 1% NP-40 along with ProteoBlock Protease inhibitor Cocktail(Fermentas, Glen Burnie, MD). The lysates were centrifuged, and equalvolumes of supernatant were boiled with 4× NuPAGE-LDS sample buffer(Invitrogen, Rockville, MD) for 5 minutes at 95° C. Proteins wereseparated using 4-12% Bis-Tris gels (Invitrogen). N-terminal c-Mycantibody (Epitomics Inc., Burlingame, CA) was used at 1:1000 to performwestern blots. Secondary anti-rabbit IgG conjugated to HRP was used at1:5000. Super Signal West Pico chemiluminescent substrate (ThermoScientific Fisher, Rockford) was used to detect bound antibodies. Forprotein normalization, the blots were stripped and reprobed using aβ-actin antibody (Sigma Aldrich, St. Louis, MO).

Undifferentiated EBs were cultured in either complete RPMI or serum-freemedia with neural growth factors for 10-15 days. Similarly, lungendothelial cells from WT and CD47-null were plated for 10-15 days withEGM2 medium at 37° C. The endothelial cells and differentiated EBs werewashed with 1×PBS, and cell lysates were made using RIPA buffer. Thelysates were centrifuged, and equal volumes of supernatant were boiledwith 4×NuPAGE-LDS sample buffer (Invitrogen) for 5 min at 95° C.Proteins were separated using 4-12% or 12% Bis-Tris gels (Invitrogen).Primary SOX2 (Abeam, Cambridge, MA), nestin (Covance, Princeton. NJ;1:500), KLF4, OCT4, SOX2 (Stemgent. Cambridge, MA), Tuj 1(Neuron-specific class III beta-tubulin, Neuromics, Edina, MN), GFAP(DAKO, Carpinteria, CA) smooth muscle actin (Sigma-Aldrich, St. Louis,MO), and AFP (Cell Signaling. Danvers, MA) antibodies were used at1:1000 to perform Western blots. Secondary anti-rabbit IgG or anti-mouseIgG conjugated to HRP were used at 1:5000. Super Signal West Picochemiluminescent substrate (Thermo Scientific Fisher) was used to detectbound antibodies. For protein normalization, the blots were reprobedusing a β-actin antibody (Sigma-Aldrich).

Single cell dilferentiation: EB-like clusters were formed using serumfree EBM media for 36 hour. A single EB-like cluster was dissociated into single cell suspension using ACCUTASE™ (BD Biosciences) celldetachment solution and was plated at limiting dilution into 96-wellplates and assessed for colony formation over 7 days. A colony waspicked, expanded and plated further in to 4-Well LabTek Chambers usingneural, smooth muscle and hepatocytes growth media. After 7 days, thecells were stained with antibodies against TUJI (ectoderm), smoothmuscle actin (mesoderm), and AFP (endoderm). WT murine lung endothelialcells were also cultured under the same conditions but were unable todifferentiate and were negative for these markers.

BrdU staining for Asymmetric cell division: Asymmetric cell division wasanalyzed as described with slight modifications. WT and CD47 null cells(passage 1) were labeled with BrdU (1uM) for 5 days and then chased inBrdU-free medium for 24 h and followed by cytochalasin B at 2 μM for 24hours. The BrdU labeled cells were fixed with 70% ethanol for 30 min.The cells were denatured with 2N HCl/0.5% Triton X-100 for 60 min. Thecells were washed in PBS/0.5% TX-100/0.1% BSA. The cells were stainedwith mouse-anti-BrdU (Calbiochem) using a dilution of 1:100 overnight at4° C. Secondary antibodies donkey-anti-mouse IgG-Alexa 594 or Alexa 488(Invitrogen) were used (1:500) for 1 hour at RT. The cells were mountedusing VECTASHIELD (Vector Laboratories). Images were acquired at 40×using an Olympus microscope. The total cells negative for BrdU andpositive for DAPI were counted manually.

Continuous growing CD47-null cells were labeled with BrdU for 10 days.One hundred percent BrdU incorporation was confirmed usingimmunofluorescent detection of nuclear BrdU labeling with confocalmicroscopy. The BrdU labeled cells were chased for 2 consecutive celldivisions in BrdU-free medium (72 hours). The mitotic cells wereobtained by gently shaking the flask. The mitotic cells were plated inglass bottom Micro Well dishes (MatTek Corporation) along withcytochalasin B for 24 hours. The cells formed EB like clusters and werestained with BrdU antibody and green fluorescent phalloidin conjugate.Images were captured using a Zeiss 780K confocal microscopy at 63×.

Transient CD47 re- or over-expression: Isolated mouse lung endothelialcells, Raji Burkitt's lymphoma cells. B16 melanoma cells, and Myc nullRat1 fibroblast cells were plated overnight in six-well plates. Thecells were transfected with CD47-FLAG expression plasmid (Kaur et al., JBiol Chem 286, 141991-15002, 2011) and/or human c-Myc-GFP plasmid(manuscript in preparation). The cells were transfected using Opti-MEM®I Reduced Serum Medium (Invitrogen) and the FuGENE® HD Transfection kit(Roche). The serum free medium was replaced with complete RPMI mediumfive hours after transfection. The cells were analyzed at 24-36 hourspost transfection. The supernatants were analyzed for lactatedehydrogenase (LDH) release using CytoTox 96® Non-RadioactiveCytotoxicity Assay (Promega Corporation, Madison, WI. USA).

JINB8 cells were transfected with CD47-V5 construct using Amaxa™Nucleofector™ (Lonza) non-viral transfection technology. Thetransfection efficiency was determined by flow Cytometry. The CD47-V5transfected cells were purified using magnetic beads bound with humanCD47 antibody (B6H12). The transfected cells were loaded onto MACScolumn which was placed in the magnetic field of a MACS® separator(Miltenyi Biotec). The magnetic bead labeled CD47⁺ cells were retainedon the column. The unlabeled cells ran through and were depleted fromCD47⁺ cells. After removal of the column, the CD47⁺ cells bound tomagnetic beads were eluted. The pure population of CD47⁺ cells wasstably cultured using G418 (250 μg/ml). The pure population of CD47 andJurkat cells were centrifuged and re-suspended in RPMI with 1% FBS at10⁶ cells/ml. Cells were plated in 12 well plates and treated with 1μg/ml (2.2 nM) thrombospondin-1, and total RNA was isolated usingTRIzol®. The relative gene expression of c-MYC was measured using GAPDHas a control.

CD47 knockdown in T cells and in WT mice: A translation-blockingantisense morpholino oligonucleotide complementary to human and murineCD47 (CGTCACAGGCAGGACCCACTGCCCA; SEQ ID NO: 35) and a 5-base mismatchcontrol morpholino (CGTgACAGcCAcGACCgACTGCgCA; SEQ ID NO: 36) wereobtained from GeneTools, LLC (Philomath, OR) as previously described(Isenberg et al., Circ Res 100, 712-720, 2007). Primary T cells isolatedfrom c-Myc EGFP knock-in mouse were transfected using morpholinos (2.5μM) along with Endo-porter (GeneTools. LLC) delivery reagent accordingto manufacturer's instructions. Mice were treated by injection of 750 μlof 10 μM morpholino in saline as described (Maxhimer et al., Sci.Transl. Med. 1, 3ra7, 2009). Organs were harvested for mRNA isolationafter 48 hours.

Modulation of stem cell transcription factors by thrombospondin-1:Jurkat and JinB8 cells were centrifuged and re-suspended in RPMI+1% FBSat 10⁶ cells/ml. Cells were plated in 12 well plates and treated with 1μg/ml (2.2 nM) TSP1 using the indicated times and concentrations, andtotal RNA was isolated using TRIzol® reagent.

Cell proliferation assays: Equal numbers of mouse lung endothelial cellsfrom WT and CD47 (Passage 1) were plated in 96 well plates using RPMI+1%FBS. After 72 hours, net cell proliferation was assessed by the increasein formazan absorbance versus controls assessed at time 0 using CellTiter 96R aqueous MTS kit (Promega, Madison, WI). DNA synthesis wasmeasured using a BrdU Assay (EMD Biosciences, Billerica, MA).

Senescence-associated f-galactosidase Assay: Senescent cells weredetected in WT and CD47 null lung endothelial cells (Passage 3) byhistological staining for a senescence-associated β-galactosidase(Debacq-Chainiaux et al., Nat Protoc 4, 1798-1806, 2009). The cells weredried, and images were taken using a phase contrast objective. Thepositive cell number was expressed as a percentage of the total cells.

Statistical Analysis: Two-way ANOVA with replication was used foranalyzing real time PCR. Student t-test was used for cell proliferation,cell cytotoxicity and senescence cell assays, which were performed intriplicate. All results are presented as mean f SD.

Results

Loss of CD47 allows self-renewal with increased c-Myc expression:Primary cells isolated from CD47-null mice exhibit a remarkableadvantage in adapting to the stress of tissue culture. Lung endothelialcells isolated from WT C57Bl/6 mice had limited survival andproliferative capacities in primary culture as assessed by reduction of[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) and bromodeoxyuridine incorporation (FIGS. 1A, B) and rapidlybecame senescent upon passage (FIG. 8A). In contrast, CD47-null lungendothelial cells at first passage showed enhanced plating efficiencyand proliferation at several cell densities (FIGS. 1A, B). Upon repeatedpassage, WT primary cells became flattened and vacuolated, whereas theCD47-null endothelial cells consistently maintained awell-differentiated cobblestone morphology for several months incontinuous culture (FIG. 8B). Continuously proliferating cell lines werereproducibly obtained when primary CD47-null cells were repeatedlypassaged but were rarely obtained from WT cultures. Similar enhancedcell culture potential was observed in CD47 null dermal papillary cellscompared to wild type cells. The CD47-null cells generally lackedexpression of the senescence-associated acidic β-galactosidase marker(Kurz et al., J. Cell Sci. 113:3613-3622, 2000) that rapidly appeared inthe WT cells (FIG. 1C). High frequency generation of continuouslyproliferating cell lines was also observed for vascular smooth musclecells and CD3⁺ T cells isolated from CD47-null mice and for lungendothelial cells cultured from mice lacking the CD47 ligandthrombospondin-1 (FIGS. 8C, D).

The ability of CD47-null and thrombospondin-1-null cells to continuouslyproliferate could reflect either increased escape from senescence orinduction of a self-renewing stem cell phenotype. Several genes havebeen identified that enable primary cells to escape senescence andbecome immortalized including p53, Rb, p16-INK4A, and c-Myc (Wang et al.Cell Cycle 10:57-67, 2011). Of these, only c-Myc mRNA levels weresignificantly elevated relative to HPRT1 mRNA levels in the primary cellcultures and remained elevated upon repeated passage (FIGS. 2A, B).Protein levels for c-Myc were also elevated in primary CD47-null lungendothelial cells as detected by Western blotting (FIGS. 2C and 3E),immunofluorescence (FIG. 2E), and flow cytometry (FIG. 2F). Most c-Mycwas nuclear, but filamentous cytoplasmic staining was also noted in theCD47-null cells, consistent with known association of c-Myc withmicrotubules (Alexandrova et al., Mol. Cell Biol. 15:5188-5195, 1995).Characteristic of a pure endothelial cell culture, the continuouslygrowing CD47-null cells uniformly expressed VEGFR2 and heterogeneouslyexpressed CD31 (Pusztaszeri et al., J. Histochem. Cytochem. 54:385-395,2006) (FIG. 3A). Absence of vascular smooth muscle cell contaminationwas indicated by the lack of detectable α-smooth muscle actinexpression, although this was detectable at low levels in WT endothelialcell cultures (FIG. 3E).

CD47 coordinately regulates stem cell transcription factors: Becauseelevated c-Myc expression also promotes self-renewal of stem cells (Kimet al., Proc. Natl. Acad Sci. USA 108:4876-4881, 2011) and is necessaryunder some conditions for embryonic stem cell self-renewal (Varlakhanovaet al., Differentiation 80:9-19, 2010), we examined the expression ofadditional transcription factors that support stem cell reprogramming(Okita and Yamanaka, Philos. Trans. R. Soc. Lond B Biol. Sci.366:2198-2207, 2011) and found significant elevation of mRNA for Sox2,Klf4, and Oct4 and the stem cell marker nestin in primary CD47-nullendothelial cells (FIG. 2D). Oct4 protein expression was detected byimmunofluorescence in a majority of the CD47 null cells, but not in WTcells (FIG. 3A). Sox2 and Klf4 were detectable in a subset of CD47-nullcells, whereas Klf4, Oct4, and c-Myc were undetectable byimmunofluorescence in WT endothelial cells and Sox2 positive cells wererarely seen (FIG. 3A). Some Sox2 staining in CD47 null cells wascytoplasmic, consistent with its reported subcellular localization inearly embryonic cells (Keramari et al., PLoS One 5:e13952, 2010), butthe majority of staining was nuclear. Western blotting confirmedelevated Klf4 and Oct4 levels in CD47-null versus WT endothelial cells(FIG. 3E). Flow cytometry also confirmed increased Oct4 expression inCD47-null cells (FIG. 3F).

These data suggested that CD47-null endothelial cell cultures contain alarger fraction of stem cells, which characteristically exhibitasymmetric cell division (Sundaraman et al., Circ. Res. 110:1169-1173,2012; Pine et al., Proc. Natl. Acad. Sci. USA 107:2195-2200, 2010). Thefrequency of asymmetric division was examined in WT and CD47-nullendothelial cell cultures uniformly labeled using BrdU by chasing withunlabeled medium for 24 hours in the presence of cytochalasin B toinhibit cytokinesis. Asymmetric division was indicated by adjacentDAPI+nuclei where only one cell retained BrdU staining (FIG. 3G, upperpanel). Such cells were significantly more abundant in the CD47-nullcultures (FIG. 3G, lower panel).

Efficient cystic embryoid body formation by CD47-null cells: Ectopicexpression of Klf4, Sox2, Oct4, and c-Myc, or ALK2 bearing an activatingmutation, in human umbilical vein endothelial cells enables efficientgeneration of multipotent or induced pluripotent stem (iPS) cells(Panopoulos et al., PLoS One 6:e19743, 2011; Medici et al., Nature Med.16:1400-1406, 2010), suggesting that self-renewal of the CD47-nullendothelial cells might arise from increased numbers of stem cells inthese cultures. Examination of characteristic stem cell markers revealedthat continuously propagated CD47-null endothelial cells expressedSca-1, and 24% of the cells were CD14⁺/CD11⁺ (FIGS. 3C and D), which arecharacteristic markers for endothelial precursor cells (Rehman et al.,Circulation 107:1164-1169, 2003). However, CD47-null endothelial cellsdid not express detectable levels of the pluripotency marker SSEA-1 orthe stem cell marker c-Kit (FIG. 3A). Because elevated expression ofc-Myc, Sox2, Oct4 and Klf4 in some types of primary cells is sufficientto induce cystic EB formation (Itskovitz-Eldor et al., Mol. Med 6:88-95,2000), we examined whether loss of CD47 circumvents the need toartificially elevate these factors for inducing EBs. Indeed, transfer ofprimary CD47-null endothelial cells or CD47-null cells growncontinuously for 6 months into serum-free medium in the absence of anyfeeder cells within 2 days induced efficient formation of floated cellaggregates that resembled EBs and continued to proliferate in this state(FIGS. 3B, 9A and B). These were never observed when WT endothelialcells were placed into the same medium, and WT cells did not survive inserum-free media. Cells in the EB-like clusters exhibited strong nuclearc-Myc staining by immunofluorescence (FIG. 3B) and flow cytometryindicated a subpopulation with stronger c-Myc expression than thatobserved in primary CD47-null endothelial cultures (compare FIG. 3H andFIG. 2F). Unlike CD47-null cells in endothelial cell medium, cells inthe EB-like clusters expressed additional stem cell markers includingalkaline phosphatase, nestin, SSEA-1 and c-Kit (FIGS. 3B, 10A-H).Consistent with their expression of stem cell markers, cells in EB-likeclusters frequently exhibited asymmetric cell division (FIG. 3I).

CD47-null cells were grown without feeder cells in ES medium containingLIF adopted colony morphologies similar to V6.5 mouse ES cells grown inthe same medium with MEF feeder cells (FIG. 3J). Immunohistochemicalanalysis of these cells in ES medium demonstrated that CD47-null andV6.5 ES cells contained similar subpopulations that expressed elevatedlevels of nuclear Oct4, Sox2, and Nanog (FIG. 3K).

Microarray analyses (GEO accession number GSE43133) were performed toglobally assess the stem cell character of CD47-null endothelial cellsand EB-like clusters derived by culture in serum-free medium for 36hours. A global principal component analysis revealed that CD47 nullendothelial cells and EB-like clusters derived from them clustered nearpublished iPS and ES cells, whereas WT cells did not. To characterizethe changes in gene expression that accompany EB-like cluster formationin the CD47 null cells, we compared global gene expression in CD47 nullEB-like clusters to that of CD47-null cells before removal of serum andendothelial growth factors and found 383 genes with significant changes.Of these, 255 clustered with genes that showed similar up- ordown-regulation in the V6.5 ES cells (FIG. 23A). A GeneSet enrichmentanalysis (GSEA) identified 39 of these up-regulated genes that areincluded in the molecular signature for human ES cells defined byBhattacharya et al. (Blood 103:2956-2964, 2004: FIG. 23B). The remaininggenes included endothelial-specific genes that were significantlydown-regulated (e.g., thrombomodulin) and epithelial/mesenchymaltransition genes that were induced when CD47-null cells formed EB-likeclusters. Notably, expression of Kdr, which encodes VEGFR2, decreased10-fold, consistent with loss of VEGFR2 immunoreactivity in theCD47-null EB-like clusters.

Deletion of CD47 permits efficient reprogramming: To determine whetherCD47-null EB-like clusters are competent to give rise to the three germlayers, 6 day-old undifferentiated EB-like clusters were plated ongelatin-coated Willico-dishes for 36 hours containing neural, smoothmuscle, or hepatocyte differentiation media (FIGS. 4A-C). Appearance ofcells expressing smooth muscle actin indicated mesoderm differentiation(FIG. 4A). Cells expressing neuron-specific βIII tubulin and glialfibrillary acidic protein (GFAP) indicated ectoderm differentiation(FIG. 4B). Cells expressing α-fetoprotein (AFP) indicated endodermdifferentiation (FIG. 4C). Because each lineage could arise fromdifferent lineage-committed stem cells in the EB-like clusters, weexpanded a single clone from CD47-null EB-like clusters and repeated theabove differentiation study. Again, cells expressing characteristicmarkers of the three embryonic lineages were obtained (FIGS. 4D-F).Therefore, the CD47-null cells are multipotent.

Morphological differentiation of EBs provides another in vitroassessment of pluripotency (Sheridan et al., Stem Cells Int.2012:738910, 2012). Differentiation of EB-like clusters in complete RPMImedium for 10-15 days resulted in morphological differentiationcharacteristic of all three embryonic germ layers (FIGS. 11A-G).Differentiation was accompanied by a loss of SSEA1 expression anddecreased expression of other stem cell markers (not shown).

These results suggested that CD47-null EB-like clusters containpluripotent cells. However, limited fibrotic responses and no teratomaformation was observed when CD47-null EB-like clusters were injectedinto NOD.Cg-Prkdcscid II2rgtm1Wjl/SzJ mice under conditions where v6.5ES cells formed teratomas. Therefore, the CD47-null EB-like clusters maynot be fully pluripotent. Alternatively, teratoma formation may havebeen prevented by SIRP-dependent clearance of the CD47-null cells.

Further evidence for multipotency was obtained when differentiatedCD47-null EBs were dispersed and cultured in neural medium on a gelatincoated substrate (FIG. 12A). Ectodermal differentiation was indicated byexpression of the neuronal markers MAP2, glial fibrillary acidic protein(GFAP), neuron-specific beta III tubulin, and the astrocyte marker S100brespectively (FIG. 12A panels d-h). Some non-adherent colonies formedfrom these cells exhibited extensive neurite formation (FIG. 12A panelsa-c).

CD47 null endothelial cells cultured in hepatocyte growth mediumdeveloped into cystic EBs and then differentiated into cells thatexpressed the hepatocyte marker α-fetoprotein (AFP, FIG. 12B, panelsa-c). The CD47-null endothelial cells from which these were derived didnot express AFP (FIG. 12B, panel d). Although, a few WT endothelialcells survived in the hepatocyte medium, EB-like clusters never formed,and no expression of AFP was observed.

WT and CD47 null endothelial cells were cultured in mesenchymal cellmedium for 10 days. Only CD47 null cells formed EB-like clusters. Somecolonies of the mesenchymal differentiated cells exhibited oil Red O⁺lipid vacuoles characteristic of adipocytes (FIG. 12B panels e-i). Wealso attempted direct transdifferentiation of CD47-null endothelialcells into mesenchymal cells. Fewer cells were oil red-positive comparedto those obtained via EBs.

Dispersion of CD47-null EBs into serum-free smooth muscle mediumcontaining platelet-derived growth factor and transforming growthfactor-β1 resulted in differentiation of cells with typical vascularsmooth muscle morphology and expressing the lineage marker smooth muscleactin (FIG. 12C).

Direct hematopoietic differentiation from cultured CD47 mil endothelialcells: Transfer of CD47-null endothelial cells into semisolid mediumcontaining hematopoietic growth factors resulted in growth of colonieswith phenotypes characteristic of myeloid (FIG. 13A) and erythroidcolonies (FIGS. 13B, C). Colonies were obtained at frequencies of2.6-8.3×10⁻⁴ from three independent CD47-null endothelial cultures,whereas no large colonies were observed in equivalent cultures of WTlung endothelial cells (FIG. 13D).

To confirm the potential of CD47-null endothelial cell cultures todifferentiate into the myeloid lineage, the cells were cultured withL929 conditioned medium as a source of macrophage colony stimulatingfactor (M-CSF) (Genovesi et al., Vet. Immunol. Immunopathol. 23:223-244,1989). After 10 days a change in cell morphology was accompanied by amarked increase in the percentage of Mac2+cells in treated CD47-nullcells compared to the same cells in endothelial growth medium (FIGS.13E-H). At the same time the treated cells showed loss of Sca-1expression (compare FIG. 3C and FIG. 13I). Expression levels of otherleukocyte and monocyte-specific markers including CD14, CD64, CD11c,Ly6C, Ly6G, CD11b, B220, and CD3e were unchanged. The cells wereconfirmed to lack CD47 expression (FIG. 13J).

Together, these results demonstrate that a population of multipotentcells is selectively maintained at high frequency in continuouslycultured CD47-null, but not WT, endothelial cells. These cells supportlong term maintenance of viable endothelial cells in medium containingendothelial growth factors, but when deprived of serum CD47-null cellsspontaneously generate cystic EBs that express pluripotency markers suchas alkaline phosphate, SSEA-1 and c-Kit. These in turn can be induced todifferentiate into cell types representative of all three embryonic germlayers when appropriate growth factors and cytokines are provided.

In vivo regulation of c-Myc and tissue stem cell abundance by CD47:Increased expression of c-Myc mRNA compared to that in WT mice wasdetected in several organs from CD47-null mice (FIG. 5A). Because thehighest elevation of c-Myc mRNA occurred in CD47-null spleen, weisolated several major cell types from this organ for further analysis.B cell (CD19⁺) and CD4⁺ T cell populations showed significantup-regulation of c-Myc mRNA, whereas CD8⁺ T cells and monocytes did not(FIG. 5B). Nestin, Sox2, KLF4, and Oct4 mRNA levels were also markedlyelevated in spleen from CD47-null mice (FIG. SC). Consistent with thelesser elevation of c-Myc mRNA levels in lung, Oct4, Sox2, and nestinmRNA levels were moderately elevated in lung, but KLF4 was not elevatedin this organ (FIG. 5D). Sox2 is normally expressed by Clara cells inconducting airways (Tompkins et al., PLoS One 4:e8248, 2009) and wassimilarly expressed in WT and CD47-null lung bronchiolar epithelium, butcells expressing higher levels of cytoplasmic Sox2 were selectivelyobserved throughout the alveolar space of the CD47-null lung (FIGS.5E-F).

The spleen of adult mice contains a pool of multipotent CD45⁻/Hox11⁺stem cells that reside in the sub-capsular red pulp and are capable ofdifferentiating into diverse lineages (Faustman, Discov. Med. 5:447-449,2005; Faustman and Davis, Int. J. Biochem. Cell Biol. 42:1576-1579,2010). Consistent with these reports, we observed a limited number ofcells with nuclear Sox2 protein expression in the sub-capsular region ofVT mouse spleen (FIG. 5G) and sparse expression of Sox2-expressing cellsin other compartments of the spleen. Similar subcapsular Sox2immunoreactivity was seen in spleen sections from CD47−/− mice, but moreextensive staining was observed in the adjacent red pulp (FIG. 5H, 13K,L). These differences in Sox2 protein expression are consistent with thewhole organ mRNA expression data and suggest that the absence of CD47increases the number of tissue resident stem cells in vivo.

CD47 expression acutely inhibits c-Myc expression: The above resultsestablish a genetic linkage between CD47, maintenance of stem cells, andc-Myc expression. To clarify this relationship, WT splenic T cells fromc-Myc-EGFP knock-in mice (Nie et al., Cell 151:68-79, 2012) were treatedwith a previously validated CD47-targeting antisense morpholino(Isenberg et al., Circ. Res. 100:712-720, 2007) and resulted in a 7-foldincrease in c-Myc mRNA level at 24 h (FIG. 6A). Intraperitonealinjection of the CD47 morpholino into WT mice significantly decreasedCD47 protein expression in vivo (FIG. 14A) and resulted in induction ofc-Myc as well Oct4 and Sox2 mRNA levels in spleen at 48 hours (FIG. 6B).

Conversely, re-expression of CD47 in CD47-null endothelial cells bytransiently transfecting a CD47 expression plasmid inhibited theirproliferation and viability (FIG. 6C), c-Myc mRNA and protein levelsfell when CD47 was re-expressed at a level sufficient to cause growthinhibition (FIGS. 6D-E. 14B-C). Growth suppression by transientlyexpressing CD47 could be bypassed by co-transfecting the cells with ac-Myc expression vector (FIG. 6C). Transient re-expression of CD47 inthe null endothelial cells also decreased mRNA levels for KLF4, Sox2,and nestin (FIG. 6F). Notably, over-expressing c-Myc alone increasednestin and Oct4 mRNA expression, but co-expression of c-Myc with CD47did not overcome the inhibitory effect of CD47 expression on KLF4, Sox2,or nestin, indicating that these CD47 signaling targets areMyc-independent.

Thrombospondin-1 controls c-Myc via CD47: The JinB8 somatic mutant ofthe Jurkat human T lymphoma cell line lacks CD47 (Reinhold et al., Int.Immunol. 11:707-718, 1999) and exhibited a similar over-expression ofc-Myc mRNA relative to the parental Jurkat cells (FIG. 7A). Therefore,CD47 also negatively regulates c-Myc expression in human cells. Micelacking the CD47 ligand thrombospondin-1 share most of the stressresistance phenotypes of CD47 null mice (Isenberg et al., Am. J. Pathol.173:1100-1112, 2008; Roberts et al., Matrix Biol. 31:162-169, 2012), andmuscle explants from thrombospondin-1-null mice exhibit increasedvascular outgrowth into three-dimensional collagen gels relative to WTexplants (Zhou et al., Oncogene 25:536-545, 2006). Consistent with theseobservations and the continuous growth of thrombospondin-1-nullendothelial cells shown in FIG. 8 , c-Myc levels in Jurkat T cells weretransiently induced but then strongly inhibited by treatment with 2.2 nMthrombospondin-1 (FIG. 7B). Likewise treating low passage human renaltubular epithelial cells with TSP1 (2.2 nM) decreased expression ofself-renewal transcription genes. Picomolar concentrations ofthrombospondin-1 were sufficient to inhibit c-Myc expression in Jurkatcells at 24 hours, but the elevated c-Myc mRNA levels in Jurkat cellslacking CD47 were not significantly inhibited by thrombospondin-1 (FIG.7C). Re-expression of CD47 in JinB8 cells reduced c-Myc mRNA expression(FIG. 7D) and restored the ability of thrombospondin-1 to inhibit c-Mycexpression (FIGS. 14D-E). Therefore, CD47 is necessary for this activityof thrombospondin-1. The transient induction of c-Myc bythrombospondin-1 may be mediated by its other receptors expressed byJurkat cells (Li et al., J. Cell Biol. 157:509-59, 2002).

Similar suppression of c-Myc levels was observed in the presence of aCD47-binding peptide derived from thrombospondin-1 (peptide 7N3. FIG.7E). A control peptide with a mutated CD47 binding motif (peptide 604)was inactive. Therefore. CD47 engagement is sufficient to inhibit c-Mycexpression without the participation of other thrombospondin-1receptors.

Endogenous thrombospondin-1 also controls expression of c-Myc mRNA invivo (FIGS. 7F, G), c-Myc mRNA levels were elevated approximately 3-foldin thrombospondin-1-null spleen and lung tissues relative to thecorresponding WT organs. Consistent with the data for CD47-null mice,Oct4, Sox2 and KLF4 mRNA levels were also elevated inthrombospondin-1-null spleen, but their levels were not significantlyincreased in lung.

Dysregulation of c-Myc confers resistance to CD47 signaling: One paradoxthat arises from the above results is that high CD47 expression appearsto be a disadvantage for cells because it suppresses c-Myc expression,yet many tumor cells and some stem cells have been reported to haveelevated CD47 expression (Chao et al., Cancer Res. 71:1374-1384, 2011;Chao el al., Cell 142:699-713, 2010: Majeti et al., Cell 138:286-299,2009; Willingham et al., Proc. Natl. Acad. Sci. USA 109:6662-6667,2012). One possible explanation is that other pathways that drive c-Mycexpression could overcome the inhibitory effects of CD47 signaling. Toexamine whether c-Myc is the primary target of CD47 signaling thatinhibits cell growth, we used Myc-null rat1 fibroblasts thatconstitutively express a tamoxifen activatable c-Myc-estrogen receptorchimeric protein (O'Connell et al., J. Biol. Chem. 278:12563-12573,2003). In contrast to cells expressing only native c-Myc controlled byits endogenous promoter, transfecting the Myc-expressing Rat1fibroblasts with the CD47 expression plasmid did not inhibit theirgrowth (FIG. 7H).

We previously reported that blocking CD47 conferred radioprotection tonormal cells and mice, but B16 melanomas grown in these mice were notprotected and instead showed enhanced radiosensitivity when CD47 wasblocked (Maxhimer et al., Sci. Transl. Med. 1:3ra7, 2009). This,combined with previous evidence that c-Myc expression is dysregulated inB16 cells (Huber et al., Br. J. Cancer 59:714-718, 1989), suggested thatCD47 signaling might not regulate c-Myc in these cells. Consistent withthis hypothesis, transiently over-expressing CD47 in B16 melanoma cellsdid not inhibit their growth (FIG. 7I).

Over-expression of CD47 also failed to inhibit growth or survival ofRaji Burkitt's lymphoma cells where c-Myc expression is driven byenhancer regions donated by the translocated immunoglobulin heavy chain(Kanda et al., J. Biol. Chem. 275:32338-32346, 2000) (FIG. 7H). Inagreement with these growth data, cell cytotoxicity (LDH release) wasincreased by re-expressing or over expressing CD47-FLAG in normal mouselung endothelial cells but not in B16 melanoma, Raji Burkitt's lymphoma,and Myc null Rat 1 fibroblasts (FIGS. 14F-H). Together, these resultsestablish that c-Myc is the dominant target of CD47 signaling forlimiting cell growth and suggest that this regulation requires 5′regions of the c-Myc gene, which are absent in Raji cells.

DISCUSSION

These results demonstrate that a population of multipotent stem cells isselectively maintained at high frequency in primary and continuouslycultured CD47-null endothelial cells. These cells support long termmaintenance of viable endothelial cells in medium containing endothelialgrowth factors, but when deprived of serum, CD47-null cellsspontaneously generate EB-like clusters that express pluripotencymarkers including alkaline phosphatase, Nanog, and SSEA-1. These in turncan be induced to differentiate into cell types representative of allthree embryonic germ layers when appropriate growth factors andcytokines are provided. In contrast to exhibiting these characteristicsof iPS cells, the CD47-null EB-like clusters did not form teratomas inmice. Consistent with their lack of teratoma formation, no loss of thetumor suppressor gene PTEN or activation of oncogenes including Ras wasfound in CD47-null EB-like clusters. Loss of PTEN has been reported toincrease teratoma formation by pluripotent stem cells (Lindgrean et al.,PLoS One 6:e16478, 2011) and others have shown that stem cells canremain pluripotent when teratoma formation is suppressed (Vazquez-Martinet al., Sci. Rep. 2:964, 2012). Thus, while it is possible that theCD47-null EB-like clusters might not be fully pluripotent, but theirlack of tumorigenicity provides an advantage for therapeuticapplications.

These data further reveal that suppression of c-Myc expression is animportant mechanism by which thrombospondin-1 signaling via CD47controls cell growth and differentiation, c-Myc is now recognized to bea universal amplifier of the expression of actively transcribed genes insomatic and embryonic stem cells (Nie et al., Cell 151:68-79, 2012), sothe ability of CD47 to control c-Myc expression identifies CD47 as cellsurface receptor that globally regulates gene expression. Combined withits specific regulation of the stem cell transcription factors Oct4,Sox2 and Klf4, CD47 limits the growth, self-renewal, and reprogrammingcapacity of primary murine cells in tissue culture. Suppression of thesemajor stem cell transcription factors by CD47 also occurs in vivo andcan be modulated by targeting CD47. A corresponding increase inabundance of tissue stem cells, suggested by the increased expression ofSox2 in several organs of CD47-null mice, may contribute to theremarkable ability of tissues in these mice and in thrombospondin-1 nullmice to recover from various injuries (Hayashi et al., Hepatologv55:1562-1573, 2012; Roberts et al., Matrix Biol. 31:162-169, 2012). Inaddition to the potential therapeutic utility of CD47 antagonists fortreating such injuries, the present data suggest that antagonists ofCD47 signaling could be used to increase the expansion of tissue stemcells for cell-based therapies and tissue engineering. CD47 antagonistscould also be used to enhance the generation of lineage-committed or iPScells and to circumvent the requirement for ectopic expression usingplasmids or integrating retroviruses encoding tumor promoting genes suchas c-Myc.

c-Myc expression greatly increases the frequency of iPS cells induced bycombined ectopic expression of Oct3/4, Sox2, and Klf4 (Varlakhanova etal., Differentiation 80:9-19, 2010). Because data provided herein showthat CD47 limits c-Myc expression and other studies have shown thatthrombospondin-1 inhibits endothelial progenitor cell function via CD47(Smadja et al., Arterioscler. Thromb. Vasc. Biol. 31:551-559, 2011), itis remarkable that CD47 expression is elevated on hematopoietic stemcells (Jaiswal et al., Cell 138:271-285, 2009). CD47 in this context wasproposed to prevent clearance of stem cells by NK cells or macrophagesexpressing the CD47 counter-receptor SIRPα (Jaiswal et al., Cell138:271-285, 2009; Kim el al., Tumour Biol. 29:28-34, 2008), but wepropose that such stem cells must adapt to the cell-autonomousinhibitory effects of high CD47 expression that suppress c-Myc and otherstem cell transcription factors, c-Myc expression is presumablymaintained through other regulatory pathways to preserve viability. Theloss of viability observed herein following the acute withdrawal ofc-Myc due to CD47-ligation or restoring CD47 expression in null cellsmay be an example of oncogene addiction in normal cells. These resultsindicate that a downward excursion of Myc must be carefully managed toprevent cell death or senescence.

Previous studies have implicated thrombospondin-1 as an inhibitor ofcertain stem cell functions but have not invoked CD47 as the relevantreceptor. Thrombospondin-1 null mice exhibited 5-fold more circulatingendothelial lineage-committed stem cells (EPCs,CD13⁺/VEGFR-2⁺/CD45⁻/CD117⁺) than WT mice (Shaked et a., Cancer Cell7:101-111, 2005). Because the elevation in EPCs was suppressed when thenull mice were treated with a drug targeting the thrombospondin receptorCD36, the increased number of EPCs was attributed to loss ofanti-angiogenic thrombospondin-1 signaling via CD36 in the null.Conversely, elevated thrombospondin-1 levels in diabetes and peripheralartery disease have been associated with suppression of vascular woundrepair mediated by EPCs (Ii et al., Circ. Res. 98:697-704, 2006; Smadjaet al., Arterioscler. Thromb. Vasc. Biol. 31:551-559, 2011). Notably,EPCs highly express CD47, and suppression of CD47 by RNAi enhanced theirproliferation and angiogenic potential (Smadja et al., Arterioscler.Thromb. Vasc. Biol. 31:551-559, 2011). The authors attributed this toincreased activity of the SDF-1/CXCR4 pathway, but the data presentedherein reveal a broader role of CD47 to limit stem cell function bysuppressing c-Myc and other stem cell transcription factors.Furthermore, because CD47-null stem cells show an enhanced capacity todifferentiate along diverse lineages, it appears that the inhibitoryfunction of CD47 in stem cell maintenance is not restricted to theendothelial lineage.

In light of the results presented herein, thrombospondin-1 and c-Myc canbe seen to form a negative feedback loop in normal cells that limits theexpression of both genes. This feedback would normally limit theexpression of inhibitory thrombospondin-1 and thereby promote tissuerenewal and regeneration. As an inhibitory cell surface receptor thatcontrols self-renewal. CD47 may be critical for understanding how themicroenvironment in the stem cell niche regulates stem celldifferentiation. Without being bound by theory, it appears that CD47 maydirectly transmit a negative signal from the environment that inhibitsself-renewal or proliferation, or lateral cross talk of CD47 withintegrins and growth factor receptors in the plasma membrane (Frazier etal., UCSD Nature Molecule Pages. doi:10.1038/mp.a002870.01 2010: Kaur etal., J. Biol. Chem. 285:38923-38932, 2010) may negatively modulate thesesignals in stem cells. The present studies identify thrombospondin-1 asa potentially key environmental signal that inhibits stem cellself-renewal via CD47.

Example 2: Efficient Neural Differentiation of CD47 Null Stem CellsOccurs without Malignant Transformation

CD47-null embryoid bodies were cultured in neural medium on gelatincoated dishes as described in Example 1 to induce neuroepithelialdifferentiation. The neuroepithelial morphology of the resulting cellsshowed that this germ layer arises efficiently from CD47-null EBs. WTcells were not capable of forming EBs or subsequent reprogramming. Theneural differentiation of the cells was further demonstrated by thepresence of neurites projecting from the monolayers (FIG. 15 ).

The neuroepithelial differentiated CD47-null cells were stained withmarkers to confirm their phenotypes and to determine whether malignanttransformation had occurred. The continuously growing CD47-null cellsexpressed the proliferation marker CDK2 but lacked over-expression ofthe transformation marker Ras. This demonstrates that blocking CD47permits self-renewal without causing malignant transformation of thecells. Similarly, mouse CD47 null induced stem cells grown in neuraldifferentiation medium maintained the neural marker N-cadherin but didnot lose expression of the tumor suppressor PTEN. Therefore, the CD47null cells are multipotent but are not transformed.

Example 3: Use of CD47 Ligands to Induce Self-Renewal in Primary HumanEndothelial Cells

This example describes use of CD47 binding peptides. CD47 antibodies(either anti-mouse or anti-human CD47), and CD47 antisense morpholino toinduce stem cell properties and enable self-renewal. Also shown isactivity to reprogram primary human endothelial cells.

Human umbilical vein endothelial cells (HUVEC), which express CD47,normally become senescent with repeated passage in tissue culture (FIG.16A). However, treatment with the CD47-binding peptide 7N3 (10 μM) orwith the function blocking anti-human CD47 antibody B6H12 (1 μg/ml)dramatically increased the sustained proliferation of these cells (FIGS.16B and C). Similar results were obtained with primary WT murine lungendothelial cells (which express CD47), using peptide 7N3 or a functionblocking anti-mouse CD47 antibody (clone 301) to treat primary mouselung endothelial cells (FIG. 17). Similar responses were seen on theTSP1 null cells, but their response was less than the WT cells. Thisdemonstrates that the ability of the CD47 binding peptide 7N3 and CD47blocking antibodies to induce self-renewal is not restricted to humancells and can be used in other mammalian species.

Temporary suppression of CD47 expression using an antisense CD47morpholino at 2.5 μM similarly enabled self-renewal in HUVEC (FIGS. 18Aand B). A second treatment using the same concentration of CD47morpholino showed an enhanced proliferative response. This demonstratesthat antisense suppression of CD47 is sufficient to induce self-renewal.Direct transfer of the HUVEC cells transfected with the CD47 morpholinointo neural differentiation medium resulted in sporadic appearance ofcells with neuronal phenotypes (FIG. 19 ). Therefore, antisensesuppression of CD47 is sufficient to induce reprogramming of primaryendothelial cells of a mesodermal lineage into an ectodermal lineage.

A new vial of HUVEC at passage 1 was thawed and split in to two 25 cm³Nunc tissue culture flasks. One flask (P1) was treated with CD47-MO (2.5μM). The other was kept as untreated. The HUVEC were transferred in tonew Nunc tissue culture flask 75 cm³ and cultured for 2 weeks using EGM2medium. After 2 weeks, the HUVEC (untreated and CD47-MO treated) wereassessed for generation of EBs, neural differentiation, andcardiomyocytes differentiation. HUVEC EBs: untreated and CD47-MO cellswere cultured in serum free medium for EB formation. CD47-mo anduntreated cells formed different phenotype cell aggregation after 3-6days. Direct Neural Differentiation media: Equal numbers of HUVEC cells(untreated and cd47-mo) were cultured with neural basal media (same formouse lung endothelial cells) for 6 days. CD47-MO treated cells survivedbetter than untreated. After 6 days, the neural basal media was replacedwith neural differentiation media (same used for mouse lung endothelialcells). None of the untreated HUVEC survived. The CD47-MO exhibitedneural phenotype and their survival was observed up to 15 days, althoughtheir numbers were low.

Proliferative capacities of the treated HUVEC (CD47 morpholino, 7N3peptide, or 604 control peptide) were assessed using MTS assays. By 6days post-treatment cells treated with the CD47 binding peptide 7N3showed enhanced proliferation, whereas control cells treated with theinactive peptide analog 604 showed decreased proliferation (FIG. 22A).At 6 days, cells treated with CD47 morpholino showed a slight but notsignificant enhancement of proliferation (FIG. 20A). However, when thecells were analyzed at 3 weeks post-treatment cells treated with CD47morpholino showed significantly increased proliferation relative tocontrol HUVEC (FIG. 20B).

Treatment of WT Jurkat T cells with the CD47 binding peptide 459 (alsoknown as peptide 4N1 with the sequence RFYVVMWK (SEQ ID NO: 37)) at 0.1μM significantly increased expression of mRNA encoding the stem celltranscription factor c-Myc at 72 hours relative to Jurkat cells treatedwith the control peptide 761 (with the sequence RFYGGMWK (SEQ ID NO:38)) (FIG. 21A). Treatment with the CD47-binding peptide 7N3(FIRVVMYEGKK; SEQ ID NO: 1) resulted in a more dramatic increase inc-Myc expression, whereas the control peptide 604 (with the sequenceFIRGGMYEGKK; SEQ ID NO: 2) did not (FIG. 21B).

Real time QPCR analysis of HUVEC after treatment with peptide 7N3 or theCD47 blocking antibody B6H12 and grown in EBM serum free (neural basalmedia) medium for 3 weeks showed high expression of mRNAs encoding c-Mycand other stem cell transcription factors including Sox2, Klf4, and Oct4and expression of the stem cell marker nestin (Table 1). Control HUVECdid not survive after 3 weeks in the same serum free medium. Therefore,transient blocking of CD47 in primary human cells is sufficient toinduce their conversion to self-renewing stem cells.

TABLE 1 Real-time PCR of HUVECs treated for three weeks with neuralmedium and 7N3 peptide or B6H12 antibody Gene Treatment C(t) c-Myc 7N330.18728 18.48635 B6H12 24.17887 24.59558 sox2 7N3 28.08806 29.00019B6H12 30.36231 31.37936 klf4 7N3 26.31469 26.16546 B6H12 26.9076128.41938 nestin 7N3 31.6104 31.32142 B6H12 32.83383 32.9545 18S RNA 7N36.115119 6.079316 B6H12 6.229277 6.21683 Oct4 7N3 34.47726 34.00265B6H12 N/A N/A

Finally, equal numbers of HUVEC (untreated and cd47-mo treated atpassage 1) were plated in 6-well plates with EGM2 media for 24 hours.The next day, EGM2 medium was replaced with cardiomyocytedifferentiation media (Millipore). The untreated HUVECs were unable tosurvive in this medium after 3 days, but the treated cells survived andunderwent differentiation (FIG. 22 ). CD47-MO cells survived up to 10days. Thus, antisense suppression of CD47 is sufficient to inducereprogramming of primary endothelial cells into cardiomyocyte lineage.

Though this example is provided using one specific primary cell type,this is exemplary only and it is expected that other obtainable primaryhuman cell types can also be used, such as skin fibroblasts, bone marrowcells, adipose tissue, mucosal tissue biopsies, umbilical cord,placenta, etc.

Example 4: Repopulation of Decellularized Tissue Matrix is Enhanced byCD47 Blockade

Self-renewal genes are upregulated in kidneys lacking CD47: Renalfailure is the leading indication for visceral organ transplantationworldwide. There are no currently successful bio-engineered renal organplatforms. CD47 controls multiple pro-survival signals in wound injury.It is not clear if this is true under basal non-injury conditions. mRNAlevels of freshly harvested kidneys were assessed from CD47+/+ andCD47−/− animals and significant increases were found in multipleself-renewal stem cell transcription factors including c-Myc, Klf4,Oct3/4 (FIG. 24 ) and Sox2. These results show that in the absence ofinjury CD47 limits self-renewal transcription factors andstem-cell/pluripotent potential and defines a role for CD47 in thisprocess. It has previously been shown that the TSP1-CD47 axis isupregulated in numerous diseases and in acute wound conditions. In acutekidney injury and pulmonary hypertension this has been most recentlybeen shown (Rogers et al., J. Am. Soc. Nephrol. 23:1538-1550, 2012;Bauer et al., Cardiovasc. Res. 93:682-693, 2012).

TSP1 inhibits self-renewal signals in primary human renal tubularepithelial cells, and CD47 blockade therapy elevates self-renewal genesin human renal cells: It is not clear what role the TSP1-CD47 signalcascade played on self-renewal signaling in specific renal cell types.We wanted to confirm in human cells that activation of CD47 inhibitsself-renewal genes. Renal tubular epithelial cells (rTEC) are a primarysource of injury in the kidney. Human rTEC were treated with a lowconcentration of exogenous TSP1 (2.2 nmol/L) and found suppression ofself-renewal genes including cMyc and Sox2 and Klf4 (FIG. 25 ).Conversely treating rTEC with a CD47 antibody that prevents TSP1activation of CD47 (anti-CD47 B6H12 antibody) elevated self-renewalgenes significantly including cMyc, Sox2 and Klf4 (FIG. 25 ) and Oct3/4and the stem cell marker nestin. Thus, in human renal cells CD47blockade increases the stem cell-pluripotent capacity of renal cells.

Restoration of decellularized trachea is enhanced in the absence ofCD47: Strategies developed to improve cell survival, engraftment, andangiogenesis of decellularized tracheal scaffolds have the potential toeliminate a major hurdle to wide spread utilization and clinicaltranslation of this approach with likely applications to bioengineeredskin, heart valves and joints. The trachea is the only vital organ notamenable in any particle fashion to transplantation. Attempts atbio-engineering tracheas have been to date fraught with complicationsand delayed healing. Furthermore, cartilage has been believed to benon-renewable. We hypothesized that activated CD47 inhibited cellself-renewal and restoration of decellularized tracheal matrixscaffolds. Eight weeks post-procedure decellularized WT tracheasorthotopically transplanted into CD47-null mice displayed modest cellrepopulation. In contrast WT scaffolds transplanted in the CD47−/−environment displayed dramatic cell repopulation and completerestoration of cartilage compared to transplants in WT animals (FIG. 26).

Blockade of CD47 signaling results in nephro-genesis in decellularizedmatrix: To further explore the implications of this in complex 3D organswe decellularized rat kidneys and implanted them in subcutaneous(ectopic) locations in mice. In some animals we blocked CD47 signaling.At 4 weeks following implantation the matrix was removed and tissuesections prepared and stained with H&E. As can been seen (FIG. 27 ), thematrix removed from animals with intact CD47 signaling showed minimalcellular repopulation. Conversely, matrix from animals with blocked CD47signaling showed complete cellular restoration and recapitulation ofnormal appearing renal tubules and glomeruli and patent blood vesselscontaining red blood cells (RBCs) (arrows) (FIG. 27 ). These datademonstrate complete and rapid restoration without over-activity and noevidence of cellular disorganization. In addition, these resultsoccurred with the matrix placed in an ectopic subcutaneous location,indicating that tissue engineering in vivo is a viable alternative withease of application. Finally the restoration of a vasculature in thematrix clears a further hurdle to the application of decellularizedscaffolds to organ and tissue engineering.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe claims. We therefore claim as our invention all that comes withinthe scope and spirit of these claims.

1. A method for inducing pluripotent or multipotent stem cells,comprising: obtaining primary cells from an animal; culturing theprimary cells; and contacting the cultured primary cells with an agentthat blocks CD47 signaling, thereby inducing pluripotent or multipotentstem cells.
 2. The method of claim 1, further comprising identifying andisolating a subset of pluripotent or multipotent stem cells that expressstem cell marker genes.
 3. The method of claim 2, wherein the stem cellmarker genes comprise at least one of c-Myc, Sox2, Klf4, or Oct4.
 4. Themethod of claim 1, further comprising culturing the primary cells inserum free media.
 5. The method of claim 1, wherein the inducedpluripotent or multipotent stem cells form embryoid bodies.
 6. Themethod of claim 1, wherein the agent that blocks CD47 signalingcomprises an anti-CD47 antibody or fragment thereof, a CD47-bindingpeptide, a CD47 antisense oligonucleotide, a CD47 morpholino, ananti-TSP1 antibody or fragment thereof, a TSP1-binding peptide, a TSP1antisense oligonucleotide, or a TSP1 morpholino.
 7. The method of claim1, wherein the agent comprises a small molecule capable of binding toCD47 or a small molecule capable of binding to TSP1.
 8. The method ofclaim 1, wherein the primary cells comprise endothelial cells,fibroblasts, hematopoietic cells, adipose cells, mucosal tissue cells,umbilical cord cells, or placenta cells.
 9. The method of claim 8,wherein the primary cells comprise human umbilical vein endothelialcells.
 10. A method for generating differentiated cells, comprising:obtaining primary cells from an animal; culturing the primary cells;contacting the cultured primary cells with an agent that blocks CD47signaling to produce contacted cells; isolating from among the contactedcells, cells that express stem cell marker genes; culturing the isolatedcells that express stem cell marker genes in serum-free media to produceinduced pluripotent or multipotent stem cells; and culturing the inducedpluripotent or multipotent stem cells in cell differentiation medium toproduce differentiated cells.
 11. The method of claim 10, wherein theinduced pluripotent or multipotent stem cells form embryoid bodies. 12.The method of claim 10, wherein culturing the induced pluripotent ormultipotent stem cells in cell differentiation medium comprisesculturing the induced pluripotent or multipotent stem cells in neuralcell differentiation medium, smooth muscle cell differentiation medium,hepatocyte cell differentiation medium, or mesenchymal celldifferentiation medium.
 13. The method of claim 1, wherein thedifferentiated cells comprise ectoderm-derived lineage cells.
 14. Themethod of claim 13, wherein the ectoderm-derived lineage cells compriseneuronal cells or astrocytes.
 15. The method of claim 1, wherein thedifferentiated cells comprise mesoderm-derived lineage cells.
 16. Themethod of claim 15, wherein the mesoderm-derived lineage cells comprisesmooth muscle cells, endothelial cells, hematopoietic cells, or myeloidcells.
 17. The method of claim 1, wherein the differentiated cellscomprise endoderm-derived lineage cells.
 18. The method of claim 17,wherein the endoderm-derived lineage cells comprise hepatocytes oradipocytes.
 19. The method of claim 1, wherein the agent that blocksCD47 signaling comprises an anti-CD47 antibody or fragment thereof, aCD47-binding peptide, a CD47 antisense oligonucleotide, a CD47morpholino, an anti-TSP1 antibody or fragment thereof, a TSP1-bindingpeptide, a TSP1 antisense oligonucleotide, or a TSP1 morpholino.
 20. Themethod of claim 1, wherein the agent comprises a small molecule capableof binding to CD47 or a small molecule capable of binding to TSP1. 21.The method of claim 1, wherein the primary cells comprise endothelialcells, fibroblasts, hematopoietic cells, adipose cells, mucosal tissuecells, umbilical cord cells, or placenta cells.
 22. The method of claim21, wherein the primary cells comprise human umbilical vein endothelialcells.
 23. A method for expanding differentiated cells, comprising:generating differentiated cells by the method of claim 10; andcontacting the differentiated cells with an agent that blocks CD47signaling, thereby expanding the differentiated cells.
 24. A method forexpanding differentiated cells, comprising contacting the differentiatedcells with an agent that blocks CD47 signaling.
 25. The method of claim1, where cells are cultured continuously with an agent that blocks CD47signaling.
 26. The method of claim 1, where cells are culturedtransiently with an agent that blocks CD47 signaling.
 27. Cells producedby the method of claim
 1. 28. A method to increase stem cell numbers inan animal comprising administering to the animal an agent that blocksCD47 signaling.
 29. The method of claim 28, wherein the administering tothe animal of an agent that blocks CD47 signaling comprises localadministration of the agent or systemic administration of the agent. 30.The method of claim 28, wherein the administering to the animal of anagent that blocks CD47 signaling comprises topical administration of theagent to the skin of the animal.
 31. The method of claim 28, wherein theagent that blocks CD47 signaling comprises an anti-CD47 antibody orfragment thereof, a CD47-binding peptide, a CD47 antisenseoligonucleotide, a CD47 morpholino, an anti-TSP1 antibody or fragmentthereof, a TSP1-binding peptide, a TSP1 antisense oligonucleotide, or aTSP1 morpholino.
 32. The method of claim 31, wherein the agent thatblocks CD47 signaling comprises a small molecule capable of binding toCD47.
 33. The method of claim 28, wherein administering to the animal anagent that blocks CD47 signaling comprises parenteral administration.34. A method of treating a disorder in a subject comprisingadministering cells produced by the method of claim 1 to the subject.35. The method of claim 34, wherein the cells are administered to thesubject parenterally.
 36. The method of claim 35, wherein the cells areadministered to the subject by transplantation.
 37. A kit, comprisingone or more of: a container containing an agent that blocks CD47signaling; a container containing a cell culture medium; and one or morecell culture dishes.
 38. The kit of claim 37, wherein the agent thatblocks CD47 signaling comprises an anti-CD47 antibody or fragmentthereof, a CD47-binding peptide, a CD47 antisense oligonucleotide, aCD47 morpholino, an anti-TSP1 antibody or fragment thereof, aTSP1-binding peptide, a TSP1 antisense oligonucleotide, or a TSP1morpholino. 39-41. (canceled)